Methods for screening for transdominant intracellular effector peptides and RNA molecules

ABSTRACT

Methods and compositions for screening for intracellular transdominant effector peptides and RNA molecules selected inside living cells from randomized pools are provided.

[0001] This is a continuation of U.S. Ser. No. 09/727,715, filed Nov.28, 2000, pending, which is a continuation of U.S. Ser. No. 08/963,368,filed Nov. 3, 1997, pending, which is a divisional of U.S. Ser. No.08/789,333, now U.S. Pat. No. 6,153,380, and a continuation of U.S. Ser.No. 08/787,738, pending, both filed Jan. 23, 1997, which are bothcontinuations-in-part of U.S. Ser. No. 08/589,109, pending and U.S. Ser.No. 08/589,911, abandoned, both filed Jan. 23, 1996.

FIELD OF THE INVENTION

[0002] The technical field of this invention is methods for screeningfor transdominant effector peptides and RNA molecules selected insideliving cells from randomized pools.

BACKGROUND OF THE INVENTION

[0003] Signaling pathways in cells often begin with an effector stimulusthat leads to a phenotypically describable change in cellularphysiology. Despite the key role intracellular signaling pathways playin disease pathogenesis, in most cases, little is understood about asignaling pathway other than the initial stimulus and the ultimatecellular response.

[0004] Historically, signal transduction has been analyzed bybiochemistry or genetics. The biochemical approach dissects a pathway ina “stepping-stone” fashion: find a molecule that acts at, or is involvedin, one end of the pathway, isolate assayable quantities and then try todetermine the next molecule in the pathway, either upstream ordownstream of the isolated one. The genetic approach is classically a“shot in the dark”: induce or derive mutants in a signaling pathway andmap the locus by genetic crosses or complement the mutation with a cDNAlibrary. Limitations of biochemical approaches include a reliance on asignificant amount of pre-existing knowledge about the constituentsunder study and the need to carry such studies out in vitro,post-mortem. Limitations of purely genetic approaches include the needto first derive and then characterize the pathway before proceeding withidentifying and cloning the gene.

[0005] Screening molecular libraries of chemical compounds for drugsthat regulate signal systems has led to important discoveries of greatclinical significance. Cyclosporin A (CsA) and FK506, for examples, wereselected in standard pharmaceutical screens for inhibition of T-cellactivation. It is noteworthy that while these two drugs bind completelydifferent cellular proteins—cyclophilin and FK506 binding protein(FKBP), respectively, the effect of either drug is virtually thesame—profound and specific suppression of T-cell activation,phenotypically observable in T cells as inhibition of mRNA productiondependent on transcription factors such as NF-AT and NF-κB. Libraries ofsmall peptides have also been successfully screened in vitro in assaysfor bioactivity. The literature is replete with examples of smallpeptides capable of modulating a wide variety of signaling pathways. Forexample, a peptide derived from the HIV-1 envelope protein has beenshown to block the action of cellular calmodulin.

[0006] A major limitation of conventional in vitro screens is delivery.While only minute amounts of an agent may be necessary to modulate aparticular cellular response, delivering such an amount to the requisitesubcellular location necessitates exposing the target cell or system torelatively massive concentrations of the agent. The effect of suchconcentrations may well mask or preclude the targeted response.

[0007] Thus, it is an object of the present invention to provide methodsand compositions for the effective introduction of random libraries intocells to screen for bioactive compounds.

[0008] Relevant Literature

[0009] Mann et al. (1983) Cell 33, 153-159, Pear et al. (1993) Proc.Natl. Acad. Sci. USA 9O(18):8392-6 and WO 94/19478 describe the BOSC andBING retroviral systems useful as delivery vectors for the disclosedmethods.

[0010] Scott and Craig (1994) Current Opinion in Biotechnology 5.40-48review random peptide libraries. Hupp et al. (1995) describe smallpeptides which activate the latent sequence-specific DNA bindingfunction of p53. Palzkill et al. (1994) report the selection offunctional signal cleavage sites from a library of random sequencesintroduced into TEM-1-lactamase.

SUMMARY OF THE INVENTION

[0011] The invention provides methods and compositions for screening fortransdominant bioactive agents such as pharmaceuticals. The inventionaccesses molecules or targets within living cells and provides for thedirect selection of those bioactive agents with desired phenotypiceffects.

[0012] In one aspect of the invention, methods for screening for atransdominant bioactive agent capable of altering the phenotype of acell are provided. The methods comprise the steps of a) introducing amolecular library of randomized candidate nucleic acids into a pluralityof cells, wherein each of said nucleic acids comprises a differentnucleotide sequence; b) screening the plurality of cells for a cellexhibiting an altered phenotype, wherein the altered phenotype is due tothe presence of a transdominant bioactive agent. The methods may alsoinclude the steps of c) isolating the cell(s) exhibiting an alteredphenotype, d) isolating a candidate nucleic acid from the cell(s).

[0013] The invention further provides methods for isolating a targetmolecule using either a candidate nucleic acid or the expression productof a candidate nucleic acid.

[0014] In an additional aspect, the candidate nucleic acids of theinvention are linked to fusion partners.

[0015] In a further aspect, the invention provides methods for screeningfor a transdominant bioactive agent capable of altering the phenotype ofa cell. The methods comprises the steps of a) introducing a molecularlibrary of randomized candidate nucleic acids into a first plurality ofcells, wherein each of the nucleic acids comprises a differentnucleotide sequence; b) contacting the first plurality of cells with asecond plurality of cells; and c) screening the second plurality ofcells for a cell exhibiting an altered phenotype.

[0016] In an additional aspect, the present invention provides molecularlibraries of retroviruses comprising different randomized nucleic acids,and cellular libraries containing the retroviral libraries.

BRIEF DESCRIPTION OF THE FIGURES

[0017]FIG. 1. Creation of a library of random peptides in a retrovirusDNA construct by PCR.

[0018]FIG. 2. Creation of a library of random peptides in a retrovirusDNA construct by primed DNA synthesis. (SEQ ID NO:1); (SEQ ID NO:2);(SEQ ID NO:3)

[0019]FIG. 3. Presentation constructs for localizing presentationstructures to specific cellular locales.

[0020]FIG. 4. Schematic of a retroviral construct.

DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides methods and compositions tocreate, effectively introduce into cells and screen compounds thataffect a signaling pathway. Little or no knowledge of the pathway isrequired, other than a presumed signaling event and an observablephysiologic change in the target cell. The disclosed methods areconceptually distinct from prior library search methods in that it is anin vivo stratagem for accessing intracellular signaling mechanisms. Theinvention also provides for the isolation of the constituents of thepathway, the tools to characterize the pathway, and lead compounds forpharmaceutical development.

[0022] The present invention provides methods for the screening ofcandidate bioactive agents which are capable of altering the phenotypeof cells containing the agents. The methods of the present inventionprovide a significant improvement over conventional screeningtechniques, as they allow the rapid screening of large numbers of randomoligonucleotides and their corresponding expression products in asingle, in vivo step. Thus, by delivering the random oligonucleotides tocells and screening the same cells, without the need to collect orsynthesize in vitro the candidate agents, highly efficient screening isaccomplished. In addition, the present methods allow screening in theabsence of significant prior characterization of the cellular defect perse.

[0023] Thus, the present invention provides methods for screeningcandidate bioactive agents for a transdominant bioactive agent capableof altering the phenotype of a cell.

[0024] By “candidate bioactive agents” or “candidate drugs” or“candidate expression products” or grammatical equivalents herein ismeant the expression product of a candidate nucleic acid which may betested for the ability to transdominantly alter the phenotype of a cell.As is described below, the candidate bioactive agents are the expressionproducts of candidate nucleic acids, and encompass several chemicalclasses, including peptides and nucleic acids such as DNA, messenger RNA(mRNA), antisense RNA, ribozyme components, etc. Thus, the candidatebioactive agents (expression products) may be either translationproducts of the candidate nucleic acids, i.e. peptides, or transcriptionproducts of the candidate nucleic acids, i.e. either DNA or RNA.

[0025] In a preferred embodiment, the candidate bioactive agents aretranslation products of the candidate nucleic acids. In this embodiment,the candidate nucleic acids are introduced into the cells, and the cellsexpress the nucleic acids to form peptides. Thus, in this embodiment,the candidate bioactive agents are peptides. Generally, peptides rangingfrom about 4 amino acids in length to about 100 amino acids may be used,with peptides ranging from about 5 to about 50 being preferred, withfrom about 5 to about 30 being particularly preferred and from about 6to about 20 being especially preferred.

[0026] In a preferred embodiment, the candidate bioactive agents aretranscription products of the candidate nucleic acids, and are thus alsonucleic acids. The transcription products may be either primarytranscripts or secondary translation products. That is, using theretroviral reverse transcriptase, primary DNA is made which are laterconverted into double stranded DNA. Additionally, using the primary DNA,RNA transcripts can be generated within the cell, including mRNA,antisense RNA and ribozymes or portions thereof.

[0027] At a minimum, the candidate bioactive agents comprise randomizedexpression products of the candidate nucleic acids. That is, everycandidate bioactive agent has a randomized portion, as defined below,that is the basis of the screening methods outlined herein. In addition,to the randomized portion, the candidate bioactive agent may alsoinclude a fusion partner.

[0028] In a preferred embodiment, the candidate bioactive agents arelinked to a fusion partner. By “fusion partner” or “functional group”herein is meant a sequence that is associated with the candidatebioactive agent, that confers upon all members of the library in thatclass a common function or ability. Fusion partners can be heterologous(i.e. not native to the host cell), or synthetic (not native to anycell). Suitable fusion partners include, but are not limited to: a)presentation structures, as defined below, which provide the candidatebioactive agents in a conformationally restricted or stable form; b)targeting sequences, defined below, which allow the localization of thecandidate bioactive agent into a subcellular or extracellularcompartment; c) rescue sequences as defined below, which allow thepurification or isolation of either the candidate bioactive agents orthe nucleic acids encoding them; d) stability sequences, which conferstability or protection from degradation to the candidate bioactiveagent or the nucleic acid encoding it, for example resistance toproteolytic degradation; e) dimerization sequences, to allow for peptidedimerization; or f) any combination of a), b), c), d), and e), as wellas linker sequences as needed.

[0029] In a preferred embodiment, the fusion partner is a presentationstructure. By “presentation structure” or grammatical equivalents hereinis meant a sequence, which, when fused to candidate bioactive agents,causes the candidate agents to assume a conformationally restrictedform. Proteins interact with each other largely through conformationallyconstrained domains. Although small peptides with freely rotating aminoand carboxyl termini can have potent functions as is known in the art,the conversion of such peptide structures into pharmacologic agents isdifficult due to the inability to predict side-chain positions forpeptidomimetic synthesis. Therefore the presentation of peptides inconformationally constrained structures will benefit both the latergeneration of pharmaceuticals and will also likely lead to higheraffinity interactions of the peptide with the target protein. This facthas been recognized in the combinatorial library generation systemsusing biologically generated short peptides in bacterial phage systems.A number of workers have constructed small domain molecules in which onemight present randomized peptide structures.

[0030] While the candidate bioactive agents may be either nucleic acidor peptides, presentation structures are preferably used with peptidecandidate agents. Thus, synthetic presentation structures, i.e.artificial polypeptides, are capable of presenting a randomized peptideas a conformationally-restricted domain. Generally such presentationstructures comprise a first portion joined to the N-terminal end of therandomized peptide, and a second portion joined to the C-terminal end ofthe peptide; that is, the peptide is inserted into the presentationstructure, although variations may be made, as outlined below. Toincrease the functional isolation of the randomized expression product,the presentation structures are selected or designed to have minimalbiologically activity when expressed in the target cell.

[0031] Preferred presentation structures maximize accessibility to thepeptide by presenting it on an exterior loop. Accordingly, suitablepresentation structures include, but are not limited to, minibodystructures, loops on beta-sheet turns and coiled-coil stem structures inwhich residues not critical to structure are randomized, zinc-fingerdomains, cysteine-linked (disulfide) structures, transglutaminase linkedstructures, cyclic peptides, B-loop structures, helical barrels orbundles, leucine zipper motifs, etc.

[0032] In a preferred embodiment, the presentation structure is acoiled-coil structure, allowing the presentation of the randomizedpeptide on an exterior loop. See, for example, Myszka et al., Biochem.33:2362-2373 (1994), hereby incorporated by reference, and FIG. 3).Using this system investigators have isolated peptides capable of highaffinity interaction with the appropriate target. In general,coiled-coil structures allow for between 6 to 20 randomized positions.

[0033] A preferred coiled-coil presentation structure is as follows:MGCAALESEVSALESEVASLESEVAALGRGDMPLAAVKSKLSAVKSKLASVKSKLAA CGPP (SEQ IDNO:4). The underlined regions represent a coiled-coil leucine zipperregion defined previously (see Martin et al., EMBO J. 13(22):5303-5309(1994), incorporated by reference). The bolded GRGDMP (SEQ ID NO:5)region represents the loop structure and when appropriately replacedwith randomized peptides (i.e. candidate bioactive agents, generallydepicted herein as (X)_(n), where X is an amino acid residue and n is aninteger of at least 5 (SEQ ID NO:98) or 6 (SEQ ID NO:80)) can be ofvariable length. The replacement of the bolded region is facilitated byencoding restriction endonuclease sites in the underlined regions, whichallows the direct incorporation of randomized oligonucleotides at thesepositions. For example, a preferred embodiment generates a XhoI site atthe double underlined LE site and a HindIII site at thedouble-underlined KL site.

[0034] In a preferred embodiment, the presentation structure is aminibody structure. A “minibody” is essentially composed of a minimalantibody complementarity region. The minibody presentation structuregenerally provides two randomizing regions that in the folded proteinare presented along a single face of the tertiary structure. See forexample Bianchi et al., J. Mol. Biol. 236(2):649-59 (1994), andreferences cited therein, all of which are incorporated by reference).Investigators have shown this minimal domain is stable in solution andhave used phage selection systems in combinatorial libraries to selectminibodies with peptide regions exhibiting high affinity, Kd=10⁻⁷, forthe pro-inflammatory cytokine IL-6.

[0035] A preferred minibody presentation structure is as follows:MGRNSQATSGFTFSHFYMEWVRGGEYIMSRHKHNKYTTEYSASVKGRYIVSRDTS QSILYLQKKKGPP(SEQ ID NO:6). The bold, underline regions are the regions which may berandomized. The italized phenylalanine must be invariant in the firstrandomizing region. The entire peptide is cloned in athree-oligonucleotide variation of the coiled-coil embodiment, thusallowing two different randomizing regions to be incorporatedsimultaneously. This embodiment utilizes non-palindromic BstXI sites onthe termini.

[0036] In a preferred embodiment, the presentation structure is asequence that contains generally two cysteine residues, such that adisulfide bond may be formed, resulting in a conformationallyconstrained sequence. This embodiment is particularly preferred whensecretory targeting sequences are used. As will be appreciated by thosein the art, any number of random sequences, with or without spacer orlinking sequences, may be flanked with cysteine residues. In otherembodiments, effective presentation structures may be generated by therandom regions themselves. For example, the random regions may be“doped” with cysteine residues which, under the appropriate redoxconditions, may result in highly crosslinked structured conformations,similar to a presentation structure. Similarly, the randomizationregions may be controlled to contain a certain number of residues toconfer β-sheet or α-helical structures.

[0037] In a preferred embodiment, the fusion partner is a targetingsequence. As will be appreciated by those in the art, the localizationof proteins within a cell is a simple method for increasing effectiveconcentration and determining function. For example, RAF1 when localizedto the mitochondrial membrane can inhibit the anti-apoptotic effect ofBCL-2. Similarly, membrane bound Sos induces Ras mediated signaling inT-lymphocytes. These mechanisms are thought to rely on the principle oflimiting the search space for ligands, that is to say, the localizationof a protein to the plasma membrane limits the search for its ligand tothat limited dimensional space near the membrane as opposed to the threedimensional space of the cytoplasm. Alternatively, the concentration ofa protein can also be simply increased by nature of the localization.Shuttling the proteins into the nucleus confines them to a smaller spacethereby increasing concentration. Finally, the ligand or target maysimply be localized to a specific compartment, and inhibitors must belocalized appropriately.

[0038] Thus, suitable targeting sequences include, but are not limitedto, binding sequences capable of causing binding of the expressionproduct to a predetermined molecule or class of molecules whileretaining bioactivity of the expression product, (for example by usingenzyme inhibitor or substrate sequences to target a class of relevantenzymes); sequences signalling selective degradation, of itself orco-bound proteins; and signal sequences capable of constitutivelylocalizing the candidate expression products to a predetermined cellularlocale, including a) subcellular locations such as the Golgi,endoplasmic reticulum, nucleus, nucleoli, nuclear membrane,mitochondria, chloroplast, secretory vesicles, lysosome, and cellularmembrane; and b) extracellular locations via a secretory signal.Particularly preferred is localization to either subcellular locationsor to the outside of the cell via secretion.

[0039] In a preferred embodiment, the targeting sequence is a nuclearlocalization signal (NLS). NLSs are generally short, positively charged(basic) domains that serve to direct the entire protein in which theyoccur to the cell's nucleus. Numerous NLS amino acid sequences have beenreported including single basic NLS's such as that of the SV40 (monkeyvirus) large T Antigen (Pro Lys Lys Lys Arg Lys Val) (SEQ ID NO:7),Kalderon (1984), et al., Cell, 39:499-509; the human retinoic acidreceptor-βnuclear localization signal (ARRRRP) (SEQ ID NO:8); NFκB p50(EEVQRKRQKL (SEQ ID NO:9); Ghosh et al., Cell 62:1019 (1990); NFκB p65(EEKRKRTYE (SEQ ID NO:10); Nolan et al., Cell 64:961 (1991); and others(see for example Boulikas, J.

[0040] Cell. Biochem. 55(1):32-58 (1994), hereby incorporated byreference) and double basic NLS's exemplified by that of the Xenopus(African clawed toad) protein, nucleoplasmin (Ala Val Lys Arg Pro AlaAla Thr Lys Lys Ala Gly Gin Ala Lys Lys Lys Lys Leu Asp) (SEQ ID NO:11),Dingwall, et al., Cell, 30:449-458, 1982 and Dingwall, et al., J. CellBiol., 107:641-849; 1988). Numerous localization studies havedemonstrated that NLSs incorporated in synthetic peptides or graftedonto reporter proteins not normally targeted to the cell nucleus causethese peptides and reporter proteins to be concentrated in the nucleus.See, for example, Dingwall, and Laskey, Ann, Rev. Cell Biol., 2:367-390,1986; Bonnerot, et al., Proc. Natl. Acad. Sci. USA, 84:6795-6799, 1987;Galileo, et al., Proc. Natl. Acad. Sci. USA, 87:458462, 1990.

[0041] In a preferred embodiment, the targeting sequence is a membraneanchoring signal sequence. This is particularly useful since manyparasites and pathogens bind to the membrane, in addition to the factthat many intracellular events originate at the plasma membrane. Thus,membrane-bound peptide libraries are useful for both the identificationof important elements in these processes as well as for the discovery ofeffective inhibitors. The invention provides methods for presenting therandomized expression product extracellularly or in the cytoplasmicspace; see FIG. 3. For extracellular presentation, a membrane anchoringregion is provided at the carboxyl terminus of the peptide presentationstructure. The randomized expression product region is expressed on thecell surface and presented to the extracellular space, such that it canbind to other surface molecules (affecting their function) or moleculespresent in the extracellular medium. The binding of such molecules couldconfer function on the cells expressing a peptide that binds themolecule. The cytoplasmic region could be neutral or could contain adomain that, when the extracellular randomized expression product regionis bound, confers a function on the cells (activation of a kinase,phosphatase, binding of other cellular components to effect function).Similarly, the randomized expression product-containing region could becontained within a cytoplasmic region, and the transmembrane region andextracellular region remain constant or have a defined function.

[0042] Membrane-anchoring sequences are well known in the art and arebased on the genetic geometry of mammalian transmembrane molecules.Peptides are inserted into the membrane based on a signal sequence(designated herein as ssTM) and require a hydrophobic transmembranedomain (herein TM). The transmembrane proteins are inserted into themembrane such that the regions encoded 5′ of the transmembrane domainare extracellular and the sequences 3′ become intracellular. Of course,if these transmembrane domains are placed 5′ of the variable region,they will serve to anchor it as an intracellular domain, which may bedesirable in some embodiments. ssTMs and TMs are known for a widevariety of membrane bound proteins, and these sequences may be usedaccordingly, either as pairs from a particular protein or with eachcomponent being taken from a different protein, or alternatively, thesequences may be synthetic, and derived entirely from consensus asartificial delivery domains.

[0043] As will be appreciated by those in the art, membrane-anchoringsequences, including both ssTM and TM, are known for a wide variety ofproteins and any of these may be used. Particularly preferredmembrane-anchoring sequences include, but are not limited to, thosederived from CD8, ICAM-2, IL-8R, CD4 and LFA-1.

[0044] Useful sequences include sequences from: 1) class I integralmembrane proteins such as IL-2 receptor beta-chain (residues 1-26 arethe signal sequence, 241-265 are the transmembrane residues; seeHatakeyama et al., Science 244:551 (1989) and von Heijne et al, Eur. J.Biochem. 174:671 (1988)) and insulin receptor beta chain (residues 1-27are the signal, 957-959 are the transmembrane domain and 960-1382 arethe cytoplasmic domain; see Hatakeyama, supra, and Ebina et al., Cell40:747 (1985)); 2) class II integral membrane proteins such as neutralendopeptidase (residues 29-51 are the transmembrane domain, 2-28 are thecytoplasmic domain; see Malfroy et al., Biochem. Biophys. Res. Commun.144:59 (1987)); 3) type III proteins such as human cytochrome P450 NF25(Hatakeyama, supra); and 4) type IV proteins such as humanP-glycoprotein (Hatakeyama, supra). Particularly preferred are CD8 andICAM-2. For example, the signal sequences from CD8 and ICAM-2 lie at theextreme 5′ end of the transcript. These consist of the amino acids 1-32in the case of CD8 (MASPLTRFLSLNLLLLGESILGSGEAKPQAP (SEQ ID NO:12);Nakauchi et al., PNAS USA 82:5126 (1985) and 1-21 in the case of ICAM-2(MSSFGYRTLTVALFTLICCPG (SEQ ID NO:13); Staunton et al., Nature (London)339:61 (1989)). These leader sequences deliver the construct to themembrane while the hydrophobic transmembrane domains, placed 3′ of therandom candidate region, serve to anchor the construct in the membrane.These transmembrane domains are encompassed by amino acids 145-195 fromCD8 (PQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSR (SEQ ID NO:14);Nakauchi, supra) and 224-256 from ICAM-2(MVIIVTVVSVLLSLFVTSVLLCFIFGQHLRQQR (SEQ ID NO:15); Staunton, supra).

[0045] Alternatively, membrane anchoring sequences include the GPIanchor, which results in a covalent bond between the molecule and thelipid bilayer via a glycosyl-phosphatidylinositol bond for example inDAF (PNKGSGTTSGTTRLLSGHTCFTLTGLLGTLVTMGLLT (SEQ ID NO:16), with thebolded serine the site of the anchor; see Homans et al., Nature333(6170):269-72 (1988), and Moran et al., J. Biol. Chem. 266:1250(1991)). In order to do this, the GPI sequence from Thy-1 can becassetted 3′ of the variable region in place of a transmembranesequence.

[0046] Similarly, myristylation sequences can serve as membraneanchoring sequences. It is known that the myristylation of c-srcrecruits it to the plasma membrane. This is a simple and effectivemethod of membrane localization, given that the first 14 amino acids ofthe protein are solely responsible for this function: MGSSKSKPKDPSQR(SEQ ID NO:17) (see Cross et al., Mol. Cell. Biol. 4(9):1834 (1984);Spencer et al., Science 262:1019-1024 (1993), both of which are herebyincorporated by reference). This motif has already been shown to beeffective in the localization of reporter genes and can be used toanchor the zeta chain of the TCR. This motif is placed 5′ of thevariable region in order to localize the construct to the plasmamembrane. Other modifications such as palmitoylation can be used toanchor constructs in the plasma membrane; for example, palmitoylationsequences from the G protein-coupled receptor kinase GRK6 sequence(LLQRLFSRQDCCGNCSDSEEELPTRL (SEQ ID NO:18), with the bold cysteinesbeing palmitoylated; Stoffel et al, J. Biol. Chem 269:27791 (1994));from rhodopsin (KQFRNCMLTSLCCGKNPLGD (SEQ ID NO:19); Barnstable et al.,J. Mol. Neurosci. 5(3):207 (1994)); and the p21 H-ras 1 protein(LNPPDESGPGCMSCKCVLS (SEQ ID NO:20); Capon et al., Nature 302:33(1983)).

[0047] In a preferred embodiment, the targeting sequence is a lysozomaltargeting sequence, including, for example, a lysosomal degradationsequence such as Lamp-2 (KFERQ (SEQ ID NO:21); Dice, Ann. N.Y. Acad.Sci. 674:58 (1992); or lysosomal membrane sequences from Lamp-1(MLIPIAGFFALAGLVLIVLIAYLIGRKRSHAGYQTI (SEQ ID NO:22), Uthayakumar etal., Cell. Mol. Biol. Res. 41:405 (1995)) or Lamp-2(LVPIAVGAALAGVLILVLLAYFIGLKHHHAGYEQF (SEQ ID NO:23), Konecki et la.,Biochem. Biophys. Res. Comm. 205:1-5 (1994), both of which show thetransmembrane domains in italics and the cytoplasmic targeting signalunderlined).

[0048] Alternatively, the targeting sequence may be a mitrochondriallocalization sequence, including mitochondrial matrix sequences (e.g.yeast alcohol dehydrogenase III; MLRTSSLFTRRVQPSLFSRNILRLQST (SEQ IDNO:24); Schatz, Eur. J. Biochem. 165:1-6 (1987)); mitochondrial innermembrane sequences (yeast cytochrome c oxidase subunit IV;MLSLRQSIRFFKPATRTLCSSRYLL (SEQ ID NO:25); Schatz, supra); mitochondrialintermembrane space sequences (yeast cytochrome c1;MFSMLSKRWAQRTLSKSFYSTATGAASKSGKLTQKLVTAGVAAAGITASTLLYADSL TAEAMTA (SEQID NO:26); Schatz, supra) or mitochondrial outer membrane sequences(yeast 70 kD outer membrane protein;MKSFITRNKTAILATVMTGTAIGAYYYYNQLQQQQQRGKK (SEQ ID NO:27); Schatz, supra).

[0049] The target sequences may also be endoplasmic reticulum sequences,including the sequences from calreticulin (KDEL (SEQ ID NO:28); Pelham,Royal Society London Transactions B; 1-10 (1992)) or adenovirus E3/19Kprotein (LYLSRRSFIDEKKMP (SEQ ID NO:29); Jackson et al., EMBO J. 9:3153(1990).

[0050] Furthermore, targeting sequences also include peroxisomesequences (for example, the peroxisome matrix sequence from Luciferase;SKL; Keller et al., PNAS USA 4:3264 (1987)); farnesylation sequences(for example, P21 H-ras 1; LNPPDESGPGCMSCKCVLS (SEQ ID NO:30), with thebold cysteine geraneygerganylated; Capon, supra); geranylgeranylationsequences (for example, protein rab-5A; LTEPTQPTRNQCCSN (SEQ ID NO:31),with the bold cysteines geranylgeranylated; Farnsworth, PNAS USA91:11963 (1994)); or destruction sequences (cyclin B1; RTALGDIGN (SEQ IDNO:32); Klotzbucher et al., EMBO J. 1:3053 (1996)).

[0051] In a preferred embodiment, the targeting sequence is a secretorysignal sequence capable of effecting the secretion of the candidatetranslation product. There are a large number of known secretory signalsequences which are placed 5′ to the variable peptide region, and arecleaved from the peptide region to effect secretion into theextracellular space. Secretory signal sequences and theirtransferability to unrelated proteins are well known, e.g., Silhavy, etal. (1985) Microbiol. Rev. 49, 398-418. This is particularly useful togenerate a peptide capable of binding to the surface of, or affectingthe physiology of, a target cell that is other than the host cell, e.g.,the cell infected with the retrovirus. In a preferred approach, a fusionproduct is configured to contain, in series, secretion signalpeptide-presentation structure-randomized expression productregion-presentation structure, see FIG. 3. In this manner, target cellsgrown in the vicinity of cells caused to express the library ofpeptides, are bathed in secreted peptide. Target cells exhibiting aphysiological change in response to the presence of a peptide, e.g., bythe peptide binding to a surface receptor or by being internalized andbinding to intracellular targets, and the secreting cells are localizedby any of a variety of selection schemes and the peptide causing theeffect determined. Exemplary effects include variously that of adesigner cytokine (i.e., a stem cell factor capable of causinghematopoietic stem cells to divide and maintain their totipotential), afactor causing cancer cells to undergo spontaneous apoptosis, a factorthat binds to the cell surface of target cells and labels themspecifically, etc.

[0052] Suitable secretory sequences are known, including signals fromIL-2 (MYRMQLLSCIALSLALVTNS (SEQ ID NO:33); Villinger et al., J. Immunol.155:3946 (1995)), growth hormone (MATGSRTSLLLAFGLLCLPWLQEGSAFPT (SEQ IDNO:34); Roskam et al., Nucleic Acids Res. 7:30 (1979)); preproinsulin(MALWMRLLPLLALLALWGPDPAAAFVN (SEQ ID NO:35); Bell et al., Nature 284:26(1980)); and influenza HA protein (MKAKLLVLLYAFVAGDQI (SEQ ID NO:36);Sekiwawa et al., PNAS 80:3563)), with cleavage between thenon-underlined-underlined junction. A particularly preferred secretorysignal sequence is the signal leader sequence from the secreted cytokineIL-4, which comprises the first 24 amino acids of IL-4 as follows:MGLTSQLLPPLFFLLACAGNFVHG (SEQ ID NO:37).

[0053] In a preferred embodiment, the fusion partner is a rescuesequence. A rescue sequence is a sequence which may be used to purify orisolate either the candidate agent or the nucleic acid encoding it.Thus, for example, peptide rescue sequences include purificationsequences such as the His₆ (SEQ ID NO:99) tag for use with Ni affinitycolumns and epitope tags for detection, immunoprecipitation or FACS(fluoroscence-activated cell sorting). Suitable epitope tags include myc(for use with the commercially available 9E10 antibody), the BSPbiotinylation target sequence of the bacterial enzyme BirA, flu tags,lacZ, and GST.

[0054] Alternatively, the rescue sequence may be a uniqueoligonucleotide sequence which serves as a probe target site to allowthe quick and easy isolation of the retroviral construct, via PCR,related techniques, or hybridization.

[0055] In a preferred embodiment, the fusion partner is a stabilitysequence to confer stability to the candidate bioactive agent or thenucleic acid encoding it. Thus, for example, peptides may be stabilizedby the incorporation of glycines after the initiation methionine (MG orMGGO), for protection of the peptide to ubiquitination as perVarshavsky's N-End Rule, thus conferring long half-life in thecytoplasm. Similarly, two prolines at the C-terminus impart peptidesthat are largely resistant to carboxypeptidase action. The presence oftwo glycines prior to the prolines impart both flexibility and preventstructure initiating events in the di-proline to be propagated into thecandidate peptide structure. Thus, preferred stability sequences are asfollows: MG(X)_(n)GGPP (SEQ ID NO:38), where X is any amino acid and nis an integer of at least four.

[0056] In one embodiment, the fusion partner is a dimerization sequence.A dimerization sequence allows the non-covalent association of onerandom peptide to another random peptide, with sufficient affinity toremain associated under normal physiological conditions. Thiseffectively allows small libraries of random peptides (for example, 10⁴)to become large libraries if two peptides per cell are generated whichthen dimerize, to form an effective library of 10⁸ (10⁴×10⁴). It alsoallows the formation of longer random peptides, if needed, or morestructurally complex random peptide molecules. The dimers may be homo-or heterodimers.

[0057] Dimerization sequences may be a single sequence thatself-aggregates, or two sequences, each of which is generated in adifferent retroviral construct. That is, nucleic acids encoding both afirst random peptide with dimerization sequence 1, and a second randompeptide with dimerization sequence 2, such that upon introduction into acell and expression of the nucleic acid, dimerization sequence 1associates with dimerization sequence 2 to form a new random peptidestructure.

[0058] Suitable dimerization sequences will encompass a wide variety ofsequences. Any number of protein-protein interaction sites are known. Inaddition, dimerization sequences may also be elucidated using standardmethods such as the yeast two hybrid system, traditional biochemicalaffinity binding studies, or even using the present methods.

[0059] The fusion partners may be placed anywhere (i.e. N-terminal,C-terminal, internal) in the structure as the biology and activitypermits.

[0060] In a preferred embodiment, the fusion partner includes a linkeror tethering sequence. Linker sequences between various targetingsequences (for example, membrane targeting sequences) and the othercomponents of the constructs (such as the randomized candidate agents)may be desirable to allow the candidate agents to interact withpotential targets unhindered. For example, when the candidate bioactiveagent is a peptide, useful linkers include glycine-serine polymers(including, for example, (GS)_(n) (SEQ ID NO:102), (GSGGS)_(n) (SEQ IDNO:39) and (GGGS)_(n) (SEQ ID NO:40), where n is an integer of at leastone), glycine-alanine polymers, alanine-serine polymers, and otherflexible linkers such as the tether for the shaker potassium channel,and a large variety of other flexible linkers, as will be appreciated bythose in the art. Glycine-serine polymers are preferred since both ofthese amino acids are relatively unstructured, and therefore may be ableto serve as a neutral tether between components. Secondly, serine ishydrophilic and therefore able to solubilize what could be a globularglycine chain. Third, similar chains have been shown to be effective injoining subunits of recombinant proteins such as single chainantibodies.

[0061] In addition, the fusion partners, including presentationstructures, may be modified, randomized, and/or matured to alter thepresentation orientation of the randomized expression product. Forexample, determinants at the base of the loop may be modified toslightly modify the internal loop peptide tertiary structure, whichmaintaining the randomized amino acid sequence.

[0062] In a preferred embodiment, combinations of fusion partners areused. Thus, for example, any number of combinations of presentationstructures, targeting sequences, rescue sequences, and stabilitysequences may be used, with or without linker sequences. As is morefully described below, using a base vector that contains a cloning sitefor receiving random and/or biased libraries, one can cassette invarious fusion partners 5′ and 3′ of the library. Table 1 outlines someof the possible combinations (without specifying the presentationstructures) as follows. Using V as the variable cloning site for therandom nucleic acid libraries, and representing each fusion partner byanother letter, (i.e. N for nuclear localization sequence) eachconstruct can be named as a string of representative letters reading 5′to 3′ read as nucleic acid or N-terminal to C-terminal read as protein,such as NV or if cloned downstream of the variable region, VN. Asimplied here, the fusion partner sequences are cloned as cassettes intosites on either side of the variable region. C is for cytoplasmic (i.e.no localization sequence), E is a rescue sequence such as the mycepitope, G is a linker sequence (G10 is a glycine-serine chain of 10amino acids, and G20 is a glycine-serine chain of 20 amino acids), M isa myristylation sequence, N is a nuclear localization sequence, ssTM isthe signal sequence for a transmembrane anchoring sequence, TM is thetransmembrane anchoring sequence, GPI is a GPI membrane anchor sequence;S is a secretory signal sequence, etc. As will be appreciated by thosein the art, any number of combinations can be made, in addition to thoselisted below. TABLE 1 cytoplasmic C V C E V C V E secreted S V S E V S VE myristylated M V M E V M E G20 V transmembrane (intracellular) ssTM VssTM V TM ssTM V E TM ssTM V G20 E TM ssTM V E transmembrane (GPIlinked) ssTM V G E TM nuclear localization N E V N V E

[0063] As will be appreciated by those in the art, these modules ofsequences can be used in a large number of combinations and variations.In addition, as discussed herein, it is possible to have more than onevariable region in a construct, either to together form a new surface orto bring two other molecules together.

[0064] In a preferred embodiment, a candidate bioactive agent linked toa presentation structure is added at the variable region cloning site,V, above. Alternatively, no presentation structure is used, giving a“free” or “non-constrained” peptide or expression product.

[0065] Preferred embodiments include the following:

[0066] a) intracellular, membrane-anchored, linked (i.e. tethered) freepeptide: MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGSGGSGSGGSGSGGSGSGGSGSGGSGGG-(X)_(n)-GGPP(SEQ ID NO:41), with the secretion signal from murine CD8 in bold, thetransmembrane region of CD8 in underline, and the linker, to provideflexibility (glycine) and solubility (serine) in italics. (X)_(n)represents the random peptide, where n is an integer greater than aboutsix. A preferred embodiment utilizing this structure utilizes biasedpeptides, as described below, for example using biased SH-3domain-binding peptide libraries in the non-constrained peptidestructures, since a number of surface receptor signaling systems employSH-3 domains as part of the signaling apparatus.

[0067] b) intracellular, membrane-anchored, linked coiled coil:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGSGGSGSGGSGSGGSGSGGSGSGGSGGGCAALESEVSALESEVASLESEVAAL-(X)_(n)-LAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ IDNO:42), with the coiled-coil structure shown in underlined italics.

[0068] c) surface-tethered extracellular, non-constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGG-(X)_(n)-GGSGGSGSGGSGSGGSGSGGSGSGGSGGGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGGPP (SEQ ID NO:43).

[0069] d) surface-tethered, extracellular constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGGCAALESEVSALESEVASLESEVAAL-(X)_(n)-LAAVKSKLSAVKSKLASVKSKLAACGGSGGSGSGGSGSGGSGSGGSGSGGSGGGPQRPEDCRPRGSVKGTGLDFACDIYIWAPLAGICVALLLSLIITLICYHSRGGPP (SEQ ID NO:44).

[0070] e) secreted, non-constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGG-(X)_(n)-GGPP (SEQ ID NO:45).

[0071] f) secreted, constrained:MRPLAGGEHTMASPLTRFLSLNLLLLGESIILGSGGGAALESEVSALESEVASLESEVAAL-(X)_(n)-LAAVKSKLSAVKSKLASVKSKLAACGPP (SEQ ID NO:46).

[0072] The candidate bioactive agents as described above are encoded bycandidate nucleic acids. By “candidate nucleic acids” herein is meant anucleic acid, generally RNA when retroviral delivery vehicles are used,which can be expressed to form candidate bioactive agents; that is, thecandidate nucleic acids encode the candidate bioactive agents and thefusion partners, if present. In addition, the candidate nucleic acidswill also generally contain enough extra sequence to effect translationor transcription, as necessary. For a peptide library, the candidatenucleic acid generally contains cloning sites which are placed to allowin frame expression of the randomized peptides, and any fusion partners,if present, such as presentation structures. For example, whenpresentation structures are used, the presentation structure willgenerally contain the initiating ATG, as a part of the parent vector.For a RNA library, the candidate nucleic acids are generally constructedwith an internal CMV promoter, tRNA promoter or cell specific promoterdesigned for immediate and appropriate expression of the RNA structureat the initiation site of RNA synthesis. The RNA is expressed anti-senseto the direction of retroviral synthesis and is terminated as known, forexample with an orientation specific terminator sequence. Interferencefrom upstream transcription is alleviated in the target cell with theself-inactivation deletion, a common feature of certain retroviralexpression systems.

[0073] Generally, the candidate nucleic acids are expressed within thecells to produce expression products of the candidate nucleic acids. Asoutlined above, the expression products include translation products,i.e. peptides, or transcription products, i.e. nucleic acid.

[0074] The candidate bioactive agents and candidate nucleic acids arerandomized, either fully randomized or they are biased in theirrandomization, e.g. in nucleotide/residue frequency generally or perposition. By “randomized” or grammatical equivalents herein is meantthat each nucleic acid and peptide consists of essentially randomnucleotides and amino acids, respectively. As is more fully describedbelow, the candidate nucleic acids which give rise to the candidateexpression products are chemically synthesized, and thus may incorporateany nucleotide at any position. Thus, when the candidate nucleic acidsare expressed to form peptides, any amino acid residue may beincorporated at any position. The synthetic process can be designed togenerate randomized nucleic acids, to allow the formation of all or mostof the possible combinations over the length of the nucleic acid, thusforming a library of randomized candidate nucleic acids.

[0075] The library should provide a sufficiently structurally diversepopulation of randomized expression products to effect aprobabilistically sufficient range of cellular responses to provide oneor more cells exhibiting a desired response. Accordingly, an interactionlibrary must be large enough so that at least one of its members willhave a structure that gives it affinity for some molecule, protein, orother factor whose activity is necessary for completion of the signalingpathway. Although it is difficult to gauge the required absolute size ofan interaction library, nature provides a hint with the immune response:a diversity of 107-108 different antibodies provides at least onecombination with sufficient affinity to interact with most potentialantigens faced by an organism. Published in vitro selection techniqueshave also shown that a library size of 10⁷ to 10⁸ issufficient to findstructures with affinity for the target. A library of all combinationsof a peptide 7 to 20 amino acids in length, such as proposed here forexpression in retroviruses, has the potential to code for 20⁷ (10⁹) to20²⁰. Thus, with libraries of 10⁷ to 10⁸ per ml of retroviral particlesthe present methods allow a “working” subset of a theoretically completeinteraction library for 7 amino acids, and a subset of shapes for the20²⁰ library. Thus, in a preferred embodiment, at least 10⁶, preferablyat least 10⁷, more preferably at least 10⁸ and most preferably at least10⁹ different expression products are simultaneously analyzed in thesubject methods. Preferred methods maximize library size and diversity.

[0076] It is important to understand that in any library system encodedby oligonucleotide synthesis one cannot have complete control over thecodons that will eventually be incorporated into the peptide structure.This is especially true in the case of codons encoding stop signals(TAA, TGA, TAG). In a synthesis with NNN as the random region, there isa {fraction (3/64)}, or 4.69%, chance that the codon will be a stopcodon. Thus, in a peptide of 10 residues, there is an unacceptable highlikelihood that 46.7% of the peptides will prematurely terminate. Forfree peptide structures this is perhaps not a problem. But for largerstructures, such as those envisioned here, such termination will lead tosterile peptide expression. To alleviate this, random residues areencoded as NNK, where K=T or G. This allows for encoding of allpotential amino acids (changing their relative representation slightly),but importantly preventing the encoding of two stop residues TAA andTGA. Thus, libraries encoding a 10 amino acid peptide will have a 15.6%chance to terminate prematurely. For candidate nucleic acids which arenot designed to result in peptide expression products, this is notnecessary.

[0077] In one embodiment, the library is fully randomized, with nosequence preferences or constants at any position. In a preferredembodiment, the library is biased. That is, some positions within thesequence are either held constant, or are selected from a limited numberof possibilities. For example, in a preferred embodiment, thenucleotides or amino acid residues are randomized within a definedclass, for example, of hydrophobic amino acids, hydrophilic residues,sterically biased (either small or large) residues, towards the creationof cysteines, for cross-linking, prolines for SH-3 domains, serines,threonines, tyrosines or histidines for phosphorylation sites, etc., orto purines, etc.

[0078] In a preferred embodiment, the bias is towards peptides ornucleic acids that interact with known classes of molecules. Forexample, when the candidate bioactive agent is a peptide, it is knownthat much of intracellular signaling is carried out via short regions ofpolypeptides interacting with other polypeptides through small peptidedomains. For instance, a short region from the HIV-1 envelopecytoplasmic domain has been previously shown to block the action ofcellular calmodulin. Regions of the Fas cytoplasmic domain, which showshomology to the mastoparan toxin from Wasps, can be limited to a shortpeptide region with death-inducing apoptotic or G protein inducingfunctions. Magainin, a natural peptide derived from Xenopus, can havepotent anti-tumour and anti-microbial activity. Short peptide fragmentsof a protein kinase C isozyme (βPKC), have been shown to block nucleartranslocation of βPKC in Xenopus oocytes following stimulation. And,short SH-3 target peptides have been used as psuedosubstrates forspecific binding to SH-3 proteins. This is of course a short list ofavailable peptides with biological activity, as the literature is densein this area. Thus, there is much precedent for the potential of smallpeptides to have activity on intracellular signaling cascades. Inaddition, agonists and antagonists of any number of molecules may beused as the basis of biased randomization of candidate bioactive agentsas well.

[0079] Thus, a number of molecules or protein domains are suitable asstarting points for the generation of biased randomized candidatebioactive agents. A large number of small molecule domains are known,that confer a common function, structure or affinity. In addition, as isappreciated in the art, areas of weak amino acid homology may havestrong structural homology. A number of these molecules, domains, and/orcorresponding consensus sequences, are known, including, but are notlimited to, SH-2 domains, SH-3 domains, Pleckstrin, death domains,protease cleavage/recognition sites, enzyme inhibitors, enzymesubstrates, Traf, etc. Similarly, there are a number of known nucleicacid binding proteins containing domains suitable for use in theinvention. For example, leucine zipper consensus sequences are known.

[0080] Where the ultimate expression product is a nucleic acid, at least10, preferably at least 12, more preferably at least 15, most preferablyat least 21 nucleotide positions need to be randomized, with morepreferable if the randomization is less than perfect. Similarly, atleast 5, preferably at least 6, more preferably at least 7 amino acidpositions need to be randomized; again, more are preferable if therandomization is less than perfect.

[0081] In a preferred embodiment, biased SH-3 domain-bindingoligonucleotides/peptides are made. SH-3 domains have been shown torecognize short target motifs (SH-3 domain-binding peptides), about tento twelve residues in a linear sequence, that can be encoded as shortpeptides with high affinity for the target SH-3 domain. Consensussequences for SH-3 domain binding proteins have been proposed. Thus, ina preferred embodiment, oligos/peptides are made with the followingbiases 1. XXXPPXPXX (SEQ ID NO:47), wherein X is a randomized residue.2. (within the positions of residue positions 11 to −2):         11  10   9   8   7   6   5   4   3   2   1 (SEQ ID NO:49) MetGlyaallaa10 aa9 aa8 aa7 Arg Pro Leu Pro Pro hyd   0  −1  −2 Pro hyd hydGly Gly Pro Pro STOP atg ggc nnk nnk nnk nnk nnk aga cct ctg cct cca sbk(SEQ ID NO:48) ggg sbk sbk gga ggc cca cct TAA1.

[0082] In this embodiment, the N-terminus flanking region is suggestedto have the greatest effects on binding affinity and is thereforeentirely randomized. “Hyd” indicates a bias toward a hydrophobicresidue, i.e.—Val, Ala, Gly, Leu, Pro, Arg. To encode a hydrophobicallybiased residue, “sbk” codon biased structure is used. Examination of thecodons within the genetic code will ensure this encodes generallyhydrophobic residues. s=g,c; b=t, g, c; v=a, g, c; m=a, c; k=t, g; n=a,t, g, c.

[0083] The candidate nucleic acids are introduced into the cells toscreen for transdominant bioactive agents capable of altering thephenotype of a cell. By “introduced into ” or grammatical equivalentsherein is meant that the nucleic acids enter the cells in a mannersuitable for subsequent expression of the nucleic acid. The method ofintroduction is largely dictated by the targeted cell type, discussedbelow. Exemplary methods include CaPO₄ precipitation, liposome fusion,lipofectin®, electroporation, viral infection, etc. The candidatenucleic acids may stably integrate into the genome of the host cell (forexample, with retroviral introduction, outlined below), or may existeither transiently or stably in the cytoplasm (i.e. through the use oftraditional plasmids, utilizing standard regulatory sequences, selectionmarkers, etc.). As many pharmaceutically important screens require humanor model mammalian cell targets, retroviral vectors capable oftransfecting such targets are preferred.

[0084] In a preferred embodiment, the candidate nucleic acids are partof a retroviral particle which infects the cells. Generally, infectionof the cells is straightforward with the application of theinfection-enhancing reagent polybrene, which is a polycation thatfacilitates viral binding to the target cell. Infection can be optimizedsuch that each cell generally expresses a single construct, using theratio of virus particles to number of cells. Infection follows a Poissondistribution.

[0085] In a preferred embodiment, the candidate nucleic acids areintroduced into the cells using retroviral vectors. Currently, the mostefficient gene transfer methodologies harness the capacity of engineeredviruses, such as retroviruses, to bypass natural cellular barriers toexogenous nucleic acid uptake. The use of recombinant retroviruses waspioneered by Richard Mulligan and David Baltimore with the Psi-2 linesand analogous retrovirus packaging systems, based on NIH 3T3 cells (seeMann et al., Cell 33:153-159 (1993), hereby incorporated by reference).Such helper-defective packaging lines are capable of producing all thenecessary trans proteins -gag, pol, and env- that are required forpackaging, processing, reverse transcription, and integration ofrecombinant genomes. Those RNA molecules that have in cis the ψpackaging signal are packaged into maturing virions. Retroviruses arepreferred for a number of reasons. First, their derivation is easy.Second, unlike Adenovirus-mediated gene delivery, expression fromretroviruses is long-term (adenoviruses do not integrate).Adeno-associated viruses have limited space for genes and regulatoryunits and there is some controversy as to their ability to integrate.Retroviruses therefore offer the best current compromise in terms oflong-term expression, genomic flexibility, and stable integration, amongother features. The main advantage of retroviruses is that theirintegration into the host genome allows for their stable transmissionthrough cell division. This ensures that in cell types which undergomultiple independent maturation steps, such as hematopoietic cellprogression, the retrovirus construct will remain resident and continueto express.

[0086] A particularly well suited retroviral transfection system isdescribed in Mann et al., supra: Pear et al., PNAS USA 90(18):8392-6(1993); Kitamura et al., PNAS USA 92:9146-9150 (1995); Kinsella et al.,Human Gene Therapy 7:1405-1413; Hofmann et al., PNAS USA 93:5185-5190;Choate et al., Human Gene Therapy 7:2247 (1996); and WO 94/19478; andreferences cited therein, all of which are incorporated by reference.

[0087] In one embodiment of the invention, the library is generated in aretrovirus DNA construct backbone, as is generally described in theexamples. Standard oligonucleotide synthesis is done to generate therandom portion of the candidate bioactive agent, using techniques wellknown in the art (see Eckstein, Oligonucleotides and Analogues, APractical Approach, IRL Press at Oxford University Press, 1991);libraries may be commercially purchased. Libraries with up to 10⁹ uniquesequences can be readily generated in such DNA backbones. Aftergeneration of the DNA library, the library is cloned into a firstprimer. The first primer serves as a “cassette”, which is inserted intothe retroviral construct. The first primer generally contains a numberof elements, including for example, the required regulatory sequences(e.g. translation, transcription, promoters, etc), fusion partners,restriction endonuclease (cloning and subcloning) sites, stop codons(preferably in all three frames), regions of complementarity for secondstrand priming (preferably at the end of the stop codon region as minordeletions or insertions may occur in the random region), etc.

[0088] A second primer is then added, which generally consists of someor all of the complementarity region to prime the first primer andoptional necessary sequences for a second unique restriction site forsubcloning. DNA polymerase is added to make double-strandedoligonucleotides. The double-stranded oligonucleotides are cleaved withthe appropriate subcloning restriction endonucleases and subcloned intothe target retroviral vectors, described below.

[0089] Any number of suitable retroviral vectors may be used. Generally,the retroviral vectors may include: selectable marker genes under thecontrol of internal ribosome entry sites (IRES), which allows forbicistronic operons and thus greatly facilitates the selection of cellsexpressing peptides at uniformly high levels; and promoters drivingexpression of a second gene, placed in sense or anti-sense relative tothe 5′ LTR. Suitable selection genes include, but are not limited to,neomycin, blastocidin, bleomycin, puromycin, and hygromycin resistancegenes, as well as self-fluorescent markers such as green fluorescentprotein, enzymatic markers such as lacZ, and surface proteins such asCD8, etc.

[0090] Preferred vectors include a vector based on the murine stem cellvirus (MSCV) (see Hawley et al., Gene Therapy 1:136 (1994)) and amodified MFG virus (Rivere et al., Genetics 92:6733 (1995)), and pBABE,outlined in the examples. A general schematic of the retroviralconstruct is depicted in FIG. 4.

[0091] The retroviruses may include inducible and constitutivepromoters. For example, there are situations wherein it is necessary toinduce peptide expression only during certain phases of the selectionprocess. For instance, a scheme to provide pro-inflammatory cytokines incertain instances must include induced expression of the peptides. Thisis because there is some expectation that over-expressedpro-inflammatory drugs might in the long-term be detrimental to cellgrowth. Accordingly, constitutive expression is undesirable, and thepeptide is only turned on during that phase of the selection processwhen the phenotype is required, and then shut the peptide down byturning off the retroviral expression to confirm the effect or ensurelong-term survival of the producer cells. A large number of bothinducible and constitutive promoters are known.

[0092] In addition, it is possible to configure a retroviral vector toallow inducible expression of retroviral inserts after integration of asingle vector in target cells; importantly, the entire system iscontained within the single retrovirus. Tet-inducible retroviruses havebeen designed incorporating the Self-Inactivating (SIN) feature of 3′LTR enhancer/promoter retroviral deletion mutant (Hoffman et al., PNASUSA 93:5185 (1996)). Expression of this vector in cells is virtuallyundetectable in the presence of tetracycline or other active analogs.However, in the absence of Tet, expression is turned on to maximumwithin 48 hours after induction, with uniform increased expression ofthe whole population of cells that harbor the inducible retrovirus,indicating that expression is regulated uniformly within the infectedcell population. A similar, related system uses a mutated TetDNA-binding domain such that it bound DNA in the presence of Tet, andwas removed in the absence of Tet. Either of these systems is suitable.

[0093] In this manner the primers create a library of fragments, eachcontaining a different random nucleotide sequence that may encode adifferent peptide. The ligation products are then transformed intobacteria, such as E. coli, and DNA is prepared from the resultinglibrary, as is generally outlined in Kitamura, PNAS USA 92:9146-9150(1995), hereby expressly incorporated by reference.

[0094] Delivery of the library DNA into a retroviral packaging systemresults in conversion to infectious virus. Suitable retroviral packagingsystem cell lines include, but are not limited to, the Bing and BOSC23cell lines described in WO 94/19478; Soneoka et al., Nucleic Acid Res.23(4):628 (1995); Finer et al., Blood 83:43 (1994); Pheonix packaginglines such as PhiNX-eco and PhiNX-ampho, described below; 292T+gag-poland retrovirus envelope; PA317; and cell lines outlined in Markowitz etal., Virology 167:400 (1988), Markowitz et al., J. Virol. 62:1120(1988), Li et al., PNAS USA 93:11658 (1996), Kinsella et al., Human GeneTherapy 7:1405 (1996), all of which are incorporated by reference.

[0095] Preferred systems include PhiNX-eco and PhiNX-ampho or similarcell lines, which are two cells lines as follows. The cell lines arebased on the BING and BOSC23 cell lines described in WO 94/19478, whichare based on the 293T cell line (a human embryonic kidney linetransformed with adenovirus E1a and carrying a temperature sensitive Tantigen co-selected with neomycin). The unique feature of this cell lineis that it is highly transfectable with either calcium phosphatemediated transfection or lipid-based transfection protocols—greater than50% of 293T cells can be transiently transfected with plasmid DNA. Thus,the cell line could be a cellular milieu in which retroviral structuralproteins and genomic viral RNA could brought together rapidly forcreation of helper-defective virus. 293T cells were therefore engineeredwith stably integrated defective constructs capable of producinggag-pol, and envelope protein for either ecotropic or amphotropicviruses. These lines were called BOSC23 and Bing, respectively. Theutility of these lines was that one could produce small amounts ofrecombinant virus transiently for use in small-scale experimentation.The lines offered advantages over previous stable systems in that viruscould be produced in days rather than months.

[0096] Two problems became apparent with these first generation linesover the two years they have been in wide use. First, gag-pol andenvelope expression was unstable and the lines required vigilantchecking for retroviral production capacity; second the structure of thevectors used for protein production were not considered fully “safe” forhelper virus production; and third, one of the lines was shown to beinadvertently carrying a hygromycin-containing retrovirus. Although theBING and BOSC23 lines are useful in the present invention, all of thesepotentially problematic issues are addressed in the PhiNXsecond-generation lines. These lines are based on 293T cells as well,with the following improvements. First, the ability to monitor gag-polproduction on a cell-by cell basis was made by introducing an IRES-CD8surface marker expression cassette downstream of the reading frame ofthe gag-pol construct (other surface markers besides CD8 are alsouseful). IRES (internal ribosome entry site) sequences allow secondaryor tertiary protein translation from a single mRNA transcript. Thus, CD8expression is a direct reflection of intracellular gag-pol and thestability of the producer cell population's ability to produce gag-polcan be readily monitored by flow cytometry. Second, for both the gag-poland envelope constructs non-Moloney promoters were used to minimizerecombination potential with introduced retroviral constructs, anddifferent promoters for gag-pol and envelope were used to minimize theirinter-recombination potential. The promoters used were CMV and RSV. Twocell lines were created, PHEONIX-ECO and PHEONIX-AMPHO. Gag-pol wasintroduced with hygromycin as the co-selectable marker and the envelopeproteins were introduced with diphtheria resistance as the co-selectablemarker. Finally, the cells were screened to find a relatively rare celltype that produced gag-pol and env in a uniform distribution, althoughthis is not required. In addition, a line termed PHEONIX-gp has beenproduced that expresses only gag-pol. This line is available for furtherpseudotyping of retroviral virions with other envelope proteins such asgibbon ape leukemia virus envelope or Vesicular Stomatitis VSV-Gprotein, Xenotropic, or retargeting envelopes can also be added.

[0097] Both PHEONIX-ECO and PHEONIX-AMPHO were tested for helper virusproduction and established as being helper-virus free. Both lines cancarry episomes for the creation of stable cell lines which can be usedto produce retrovirus. Both lines are readily testable by flow cytometryfor stability of gag-pol (CD8) and envelope expression; after severalmonths of testing the lines appear stable, and do not demonstrate lossof titre as did the first-generation lines BOSC23 and Bing (partly dueto the choice of promoters driving expression of gag-pol and envelope).Both lines can also be used to transiently produce virus in a few days.Thus, these new lines are fully compatible with transient, episomalstable, and library generation for retroviral gene transfer experiments.Finally, the titres produced by these lines have been tested. Usingstandard polybrene-enhanced retroviral infection, titres approaching orabove 10⁷ per ml were observed for both PHEONIX-eco and PHEONIX-amphowhen carrying episomal constructs. When transiently produced virus ismade, titres are usually ½ to ⅓ that value.

[0098] These lines are helper-virus free, carry episomes for long-termstable production of retrovirus, stably produce gag-pol and env, and donot demonstrate loss of viral titre over time. In addition, PhiNX-ecoand PhiNX-ampho are capable of producing titres approaching or above 10⁷per ml when carrying episomal constructs, which, with concentration ofvirus, can be enhanced to 10⁸ to 10⁹ per ml.

[0099] In a preferred embodiment, the cell lines disclosed above, andthe other methods for producing retrovirus, are useful for production ofvirus by transient transfection. The virus can either be used directlyor be used to infect another retroviral producer cell line for“expansion” of the library.

[0100] Concentration of virus may be done as follows. Generally,retroviruses are titred by applying retrovirus-containing supernatantonto indicator cells, such as NIH3T3 cells, and then measuring thepercentage of cells expressing phenotypic consequences of infection. Theconcentration of the virus is determined by multiplying the percentageof cells infected by the dilution factor involved, and taking intoaccount the number of target cells available to obtain a relative titre.If the retrovirus contains a reporter gene, such as lacZ, theninfection, integration, and expression of the recombinant virus ismeasured by histological staining for lacZ expression or by flowcytometry (FACS). In general, retroviral titres generated from even thebest of the producer cells do not exceed 10⁷ per ml, unlessconcentration by relatively expensive or exotic apparatus. However, asit has been recently postulated that since a particle as large as aretrovirus will not move very far by brownian motion in liquid, fluiddynamics predicts that much of the virus never comes in contact with thecells to initiate the infection process. However, if cells are grown orplaced on a porous filter and retrovirus is allowed to move past cellsby gradual gravitometric flow, a high concentration of virus aroundcells can be effectively maintained at all times. Thus, up to a ten-foldhigher infectivity by infecting cells on a porous membrane and allowingretrovirus supernatant to flow past them has been seen. This shouldallow titres of 10⁹ after concentration.

[0101] The candidate nucleic acids, as part of the retroviral construct,are introduced into the cells to screen for transdominant bioactiveagents capable of altering the phenotype of a cell.

[0102] As will be appreciated by those in the art, the type of cellsused in the present invention can vary widely. Basically, any mammaliancells may be used, with mouse, rat, primate and human cells beingparticularly preferred, although as will be appreciated by those in theart, modifications of the system by pseudotyping allows all eukaryoticcells to be used, preferably higher eukaryotes. As is more fullydescribed below, a screen will be set up such that the cells exhibit aselectable phenotype in the presence of a bioactive agent. As is morefully described below, cell types implicated in a wide variety ofdisease conditions are particularly useful, so long as a suitable screenmay be designed to allow the selection of cells that exhibit an alteredphenotype as a consequence of the presence of a transdominant bioactiveagent within the cell.

[0103] Accordingly, suitable cell types include, but are not limited to,tumor cells of all types (particularly melanoma, myeloid leukemia,carcinomas of the lung, breast, ovaries, colon, kidney, prostate,pancreas and testes), cardiomyocytes, endothelial cells, epithelialcells, lymphocytes (T-cell and B cell) , mast cells, eosinophils,vascular intimal cells, hepatocytes, leukocytes including mononuclearleukocytes, stem cells such as haemopoetic, neural, skin, lung, kidney,liver and myocyte stem cells (for use in screening for differentiationand de-differentiation factors), osteoclasts, chondrocytes and otherconnective tissue cells, keratinocytes, melanocytes, liver cells, kidneycells, and adipocytes. Suitable cells also include known research cells,including, but not limited to, Jurkat T cells, NIH3T3 cells, CHO, Cos,etc. See the ATCC cell line catalog, hereby expressly incorporated byreference.

[0104] In one embodiment, the cells may be genetically engineered, thatis, contain erogenous nucleic acid, for example, to contain targetmolecules.

[0105] In a preferred embodiment, a first plurality of cells isscreened. That is, the cells into which the candidate nucleic acids areintroduced are screened for an altered phenotype. Thus, in thisembodiment, the effect of the transdominant bioactive agent is seen inthe same cells in which it is made; i.e. an autocrine effect.

[0106] By a “plurality of cells” herein is meant roughly from about 10³cells to 10⁸ or 10⁹, with from 10⁶ to 10⁸ being preferred. Thisplurality of cells comprises a cellular library, wherein generally eachcell within the library contains a member of the retroviral molecularlibrary, i.e. a different candidate nucleic acid, although as will beappreciated by those in the art, some cells within the library may notcontain a retrovirus, and some may contain more than one. When methodsother than retroviral infection are used to introduce the candidatenucleic acids into a plurality of cells, the distribution of candidatenucleic acids within the individual cell members of the cellular librarymay vary widely, as it is generally difficult to control the number ofnucleic acids which enter a cell during electroporation, etc.

[0107] In a preferred embodiment, the candidate nucleic acids areintroduced into a first plurality of cells, and the effect of thecandidate bioactive agents is screened in a second or third plurality ofcells, different from the first plurality of cells, i.e. generally adifferent cell type. That is, the effect of the transdominant bioactiveagents is due to an extracellular effect on a second cell; i.e. anendocrine or paracrine effect. This is done using standard techniques.The first plurality of cells may be grown in or on one media, and themedia is allowed to touch a second plurality of cells, and the effectmeasured. Alternatively, there may be direct contact between the cells.Thus, “contacting” is functional contact, and includes both direct andindirect. In this embodiment, the first plurality of cells may or maynot be screened.

[0108] If necessary, the cells are treated to conditions suitable forthe expression of the candidate nucleic acids (for example, wheninducible promoters are used), to produce the candidate expressionproducts, either translation or transcription products.

[0109] Thus, the methods of the present invention comprise introducing amolecular library of randomized candidate nucleic acids into a pluralityof cells, a cellular library. Each of the nucleic acids comprises adifferent, generally randomized, nucleotide sequence. The plurality ofcells is then screened, as is more fully outlined below, for a cellexhibiting an altered phenotype. The altered phenotype is due to thepresence of a transdominant bioactive agent.

[0110] By “altered phenotype” or “changed physiology” or othergrammatical equivalents herein is meant that the phenotype of the cellis altered in some way, preferably in some detectable and/or measurableway. As will be appreciated in the art, a strength of the presentinvention is the wide variety of cell types and potential phenotypicchanges which may be tested using the present methods. Accordingly, anyphenotypic change which may be observed, detected, or measured may bethe basis of the screening methods herein. Suitable phenotypic changesinclude, but are not limited to: gross physical changes such as changesin cell morphology, cell growth, cell viability, adhesion to substratesor other cells, and cellular density; changes in the expression of oneor more RNAs, proteins, lipids, hormones, cytokines, or other molecules;changes in the equilibrium state (i.e. half-life) or one or more RNAs,proteins, lipids, hormones, cytokines, or other molecules; changes inthe localization of one or more RNAs, proteins, lipids, hormones,cytokines, or other molecules; changes in the bioactivity or specificactivity of one or more RNAs, proteins, lipids, hormones, cytokines,receptors, or other molecules; changes in the secretion of ions,cytokines, hormones, growth factors, or other molecules; alterations incellular membrane potentials, polarization, integrity or transport;changes in infectivity, susceptibility, latency, adhesion, and uptake ofviruses and bacterial pathogens; etc. By “capable of altering thephenotype” herein is meant that the bioactive agent can change thephenotype of the cell in some detectable and/or measurable way.

[0111] The altered phenotype may be detected in a wide variety of ways,as is described more fully below, and will generally depend andcorrespond to the phenotype that is being changed. Generally, thechanged phenotype is detected using, for example: microscopic analysisof cell morphology; standard cell viability assays, including bothincreased cell death and increased cell viability, for example, cellsthat are now resistant to cell death via virus, bacteria, or bacterialor synthetic toxins; standard labeling assays such as fluorometricindicator assays for the presence or level of a particular cell ormolecule, including FACS or other dye staining techniques; biochemicaldetection of the expression of target compounds after killing the cells;etc. In some cases, as is more fully described herein, the alteredphenotype is detected in the cell in which the randomized nucleic acidwas introduced; in other embodiments, the altered phenotype is detectedin a second cell which is responding to some molecular signal from thefirst cell.

[0112] An altered phenotype of a cell indicates the presence of atransdominant bioactive agent. By “transdominant” herein is meant thatthe bioactive agent indirectly causes the altered phenotype by acting ona second molecule, which leads to an altered phenotype. That is, atransdominant expression product has an effect that is not in cis, i.e.,a trans event as defined in genetic terms or biochemical terms. Atransdominant effect is a distinguishable effect by a molecular entity(i.e., the encoded peptide or RNA) upon some separate anddistinguishable target; that is, not an effect upon the encoded entityitself. As such, transdominant effects include many well-known effectsby pharmacologic agents upon target molecules or pathways in cells orphysiologic systems; for instance, the β-lactam antibiotics have atransdominant effect upon peptidoglycan synthesis in bacterial cells bybinding to penicillin binding proteins and disrupting their functions.An exemplary transdominant effect by a peptide is the ability to inhibitNF-κB signaling by binding to IkB-α at a region critical for itsfunction, such that in the presence of sufficient amounts of the peptide(or molecular entity), the signaling pathways that normally lead to theactivation of NF-κB through phosphorylation and/or degradation of IκB-αare inhibited from acting at IκB-α because of the binding of the peptideor molecular entity. In another instance, signaling pathways that arenormally activated to secrete IgE are inhibited in the presence ofpeptide. Or, signaling pathways in adipose tissue cells, normallyquiescent, are activated to metabolize fat. Or, in the presence of apeptide, intracellular mechanisms for the replication of certainviruses, such as HIV-I, or Herpes viridae family members, or RespiratorySyncytial Virus, for example, are inhibited.

[0113] A transdominant effect upon a protein or molecular pathway isclearly distinguishable from randomization, change, or mutation of asequence within a protein or molecule of known or unknown function toenhance or diminish a biochemical ability that protein or moleculealready manifests. For instance, a protein that enzymatically cleavesβ-lactam antibiotics, β-lactamase, could be enhanced or diminished inits activity by mutating sequences internal to its structure thatenhance or diminish the ability of this enzyme to act upon and cleaveβ-lactam antibiotics. This would be called a cis mutation to theprotein. The effect of this protein upon β-lactam antibiotics is anactivity the protein already manifests, to a distinguishable degree.Similarly, a mutation in the leader sequence that enhanced the export ofthis protein to the extracellular spaces wherein it might encounterβ-lactam molecules more readily, or a mutation within the sequence thatenhance the stability of the protein, would be termed cis mutations inthe protein. For comparison, a transdominant effector of this proteinwould include an agent, independent of the β-lactamase, that bound tothe β-lactamase in such a way that it enhanced or diminished thefunction of the β-lactamase by virtue of its binding to β-lactamase.

[0114] In general, cis-effects are effects within molecules whereinelements that are interacting are covalently joined to each otheralthough these elements might individually manifest themselves asseparable domains. Trans-effects (transdominant in that under somecellular conditions the desired effect is manifested) are those effectsbetween distinct molecular entities, such that molecular entity A, notcovalently linked to molecular entity B, binds to or otherwise has aneffect upon the activities of entity B. As such, most knownpharmacological agents are transdominant effectors.

[0115] In a preferred embodiment, once a cell with an altered phenotypeis detected, the cell is isolated from the plurality which do not havealtered phenotypes. This may be done in any number of ways, as is knownin the art, and will in some instances depend on the assay or screen.Suitable isolation techniques include, but are not limited to, FACS,lysis selection using complement, cell cloning, scanning by Fluorimager,expression of a “survival” protein, induced expression of a cell surfaceprotein or other molecule that can be rendered fluorescent or taggablefor physical isolation; expression of an enzyme that changes anon-fluorescent molecule to a fluorescent one; overgrowth against abackground of no or slow growth; death of cells and isolation of DNA orother cell vitality indicator dyes, etc.

[0116] In a preferred embodiment, the candidate nucleic acid and/or thebioactive agent is isolated from the positive cell. This may be done ina number of ways. In a preferred embodiment, primers complementary toDNA regions common to the retroviral constructs, or to specificcomponents of the library such as a rescue sequence, defined above, areused to “rescue” the unique random sequence. Alternatively, thebioactive agent is isolated using a rescue sequence. Thus, for example,rescue sequences comprising epitope tags or purification sequences maybe used to pull out the bioactive agent, using immunoprecipitation oraffinity columns. In some instances, as is outlined below, this may alsopull out the primary target molecule, if there is a sufficiently strongbinding interaction between the bioactive agent and the target molecule.Alternatively, the peptide may be detected using mass spectroscopy.

[0117] Once rescued, the sequence of the bioactive agent and/orbioactive nucleic acid is determined. This information can then be usedin a number of ways.

[0118] In a preferred embodiment, the bioactive agent is resynthesizedand reintroduced into the target cells, to verify the effect. This maybe done using retroviruses, or alternatively using fusions to the HIV-1Tat protein, and analogs and related proteins, which allows very highuptake into target cells. See for example, Fawell et al., PNAS USA91:664 (1994); Frankel et al., Cell 55:1189 (1988); Savion et al., J.Biol. Chem. 256:1149 (1981); Derossi et al., J. Biol. Chem. 269:10444(1994); and Baldin et al., EMBO J. 9:1511 (1990), all of which areincorporated by reference.

[0119] In a preferred embodiment, the sequence of a bioactive agent isused to generate more candidate bioactive agents. For example, thesequence of the bioactive agent may be the basis of a second round of(biased) randomization, to develop bioactive agents with increased oraltered activities. Alternatively, the second round of randomization maychange the affinity of the bioactive agent. Furthermore, it may bedesirable to put the identified random region of the bioactive agentinto other presentation structures, or to alter the sequence of theconstant region of the presentation structure, to alter theconformation/shape of the bioactive agent. It may also be desirable to“walk” around a potential binding site, in a manner similar to themutagenesis of a binding pocket, by keeping one end of the ligand regionconstant and randomizing the other end to shift the binding of thepeptide around.

[0120] In a preferred embodiment, either the bioactive agent or thebioactive nucleic acid encoding it is used to identify target molecules,i.e. the molecules with which the bioactive agent interacts. As will beappreciated by those in the art, there may be primary target molecules,to which the bioactive agent binds or acts upon directly, and there maybe secondary target molecules, which are part of the signalling pathwayaffected by the bioactive agent; these might be termed “validatedtargets”.

[0121] In a preferred embodiment, the bioactive agent is used to pullout target molecules. For example, as outlined herein, if the targetmolecules are proteins, the use of epitope tags or purificationsequences can allow the purification of primary target molecules viabiochemical means (co-immunoprecipitation, affinity columns, etc.).Alternatively, the peptide, when expressed in bacteria and purified, canbe used as a probe against a bacterial cDNA expression library made frommRNA of the target cell type. Or, peptides can be used as “bait” ineither yeast or mammalian two or three hybrid systems. Such interactioncloning approaches have been very useful to isolate DNA-binding proteinsand other interacting protein components. The peptide(s) can be combinedwith other pharmacologic activators to study the epistatic relationshipsof signal transduction pathways in question. It is also possible tosynthetically prepare labeled peptide bioactive agent and use it toscreen a cDNA library expressed in bacteriophage for those cDNAs whichbind the peptide. Furthermore, it is also possible that one could usecDNA cloning via retroviral libraries to “complement” the effect inducedby the peptide. In such a strategy, the peptide would be required to bestochiometrically titrating away some important factor for a specificsignaling pathway. If this molecule or activity is replenished byover-expression of a cDNA from within a cDNA library, then one can clonethe target. Similarly, cDNAs cloned by any of the above yeast orbacteriophage systems can be reintroduced to mammalian cells in thismanner to confirm that they act to complement function in the system thepeptide acts upon.

[0122] Once primary target molecules have been identified, secondarytarget molecules may be identified in the same manner, using the primarytarget as the “bait”. In this manner, signalling pathways may beelucidated. Similarly, bioactive agents specific for secondary targetmolecules may also be discovered, to allow a number of bioactive agentsto act on a single pathway, for example for combination therapies.

[0123] The screening methods of the present invention may be useful toscreen a large number of cell types under a wide variety of conditions.Generally, the host cells are cells that are involved in disease states,and they are tested or screened under conditions that normally result inundesirable consequences on the cells. When a suitable bioactive agentis found, the undesirable effect may be reduced or eliminated.Alternatively, normally desirable consequences may be reduced oreliminated, with an eye towards elucidating the cellular mechanismsassociated with the disease state or signalling pathway.

[0124] In a preferred embodiment, the present methods are useful incancer applications. The ability to rapidly and specifically kill tumorcells is a cornerstone of cancer chemotherapy. In general, using themethods of the present invention, random libraries can be introducedinto any tumor cell (primary or cultured), and peptides identified whichby themselves induce apoptosis, cell death, loss of cell division ordecreased cell growth. This may be done de novo, or by biasedrandomization toward known peptide agents, such as angiostatin, whichinhibits blood vessel wall growth. Alternatively, the methods of thepresent invention can be combined with other cancer therapeutics (e.g.drugs or radiation) to sensitize the cells and thus induce rapid andspecific apoptosis, cell death, loss of cell division or decreased cellgrowth after exposure to a secondary agent. Similarly, the presentmethods may be used in conjunction with known cancer therapeutics toscreen for agonists to make the therapeutic more effective or lesstoxic. This is particularly preferred when the chemotherapeutic is veryexpensive to produce such as taxol.

[0125] Known oncogenes such as v-Abl, v-Src, v-Ras, and others, induce atransformed phenotype leading to abnormal cell growth when transfectedinto certain cells. This is also a major problem with micro-metastases.Thus, in a preferred embodiment, non-transformed cells can betransfected with these oncogenes, and then random libraries introducedinto these cells, to select for bioactive agents which reverse orcorrect the transformed state. One of the signal features of oncogenetransformation of cells is the loss of contact inhibition and theability to grow in soft-agar. When transforming viruses are constructedcontaining v-Abl, v-Src, or v-Ras in IRES-puro retroviral vectors,infected into target 3T3 cells, and subjected to puromycin selection,all of the 3T3 cells hyper-transform and detach from the plate. Thecells may be removed by washing with fresh medium. This can serve as thebasis of a screen, since cells which express a bioactive agent willremain attached to the plate and form colonies.

[0126] Similarly, the growth and/or spread of certain tumor types isenhanced by stimulatory responses from growth factors and cytokines(PDGF, EGF, Heregulin, and others) which bind to receptors on thesurfaces of specific tumors. In a preferred embodiment, the methods ofthe invention are used to inhibit or stop tumor growth and/or spread, byfinding bioactive agents capable of blocking the ability of the growthfactor or cytokine to stimulate the tumor cell. The introduction ofrandom libraries into specific tumor cells with the addition of thegrowth factor or cytokine, followed by selection of bioactive agentswhich block the binding, signaling, phenotypic and/or functionalresponses of these tumor cells to the growth factor or cytokine inquestion.

[0127] Similarly, the spread of cancer cells (invasion and metastasis)is a significant problem limiting the success of cancer therapies. Theability to inhibit the invasion and/or migration of specific tumor cellswould be a significant advance in the therapy of cancer. Tumor cellsknown to have a high metastatic potential (for example, melanoma, lungcell carcinoma, breast and ovarian carcinoma) can have random librariesintroduced into them, and peptides selected which in a migration orinvasion assay, inhibit the migration and/or invasion of specific tumorcells. Particular applications for inhibition of the metastaticphenotype, which could allow a more specific inhibition of metastasis,include the metastasis suppressor gene NM23, which codes for adinucleoside diphosphate kinase. Thus intracellular peptide activatorsof this gene could block metastasis, and a screen for its upregulation(by fusing it to a reporter gene) would be of interest. Many oncogenesalso enhance metastasis. Peptides which inactivate or counteract mutatedRAS oncogenes, v-MOS, v-RAF, A-RAF, v-SRC, v-FES, and v-FMS would alsoact as anti-metastatics. Peptides which act intracellularly to block therelease of combinations of proteases required for invasion, such as thematrix metalloproteases and urokinase, could also be effectiveantimetastatics.

[0128] In a preferred embodiment, the random libraries of the presentinvention are introduced into tumor cells known to have inactivatedtumor suppressor genes, and successful reversal by either reactivationor compensation of the knockout would be screened by restoration of thenormal phenotype. A major example is the reversal of p53-inactivatingmutations, which are present in 50% or more of all cancers. Since p53'sactions are complex and involve its action as a transcription factor,there are probably numerous potential ways a peptide or small moleculederived from a peptide could reverse the mutation. One example would beupregulation of the immediately downstream cyclin-dependent kinasep21CIP1/WAF1. To be useful such reversal would have to work for many ofthe different known p53 mutations. This is currently being approached bygene therapy; one or more small molecules which do this might bepreferable.

[0129] Another example involves screening of bioactive agents whichrestore the constitutive function of the brca-1 or brca-2 genes, andother tumor suppressor genes important in breast cancer such as theadenomatous polyposis coli gene (APC) and the Drosophila discs-largegene (Dlg), which are components of cell-cell junctions. Mutations ofbrca-1 are important in hereditary ovarian and breast cancers, andconstitute an additional application of the present invention.

[0130] In a preferred embodiment, the methods of the present inventionare used to create novel cell lines from cancers from patients. Aretrovirally delivered short peptide which inhibits the final commonpathway of programmed cell death should allow for short- and possiblylong-term cell lines to be established. Conditions of in vitro cultureand infection of human leukemia cells will be established. There is areal need for methods which allow the maintenance of certain tumor cellsin culture long enough to allow for physiological and pharmacologicalstudies. Currently, some human cell lines have been established by theuse of transforming agents such as Epstein-Barr virus that considerablyalters the existing physiology of the cell. On occasion, cells will growon their own in culture but this is a random event. Programmed celldeath (apoptosis) occurs via complex signaling pathways within cellsthat ultimately activate a final common pathway producing characteristicchanges in the cell leading to a non-inflammatory destruction of thecell. It is well known that tumor cells have a high apoptotic index, orpropensity to enter apoptosis in vivo. When cells are placed in culture,the in vivo stimuli for malignant cell growth are removed and cellsreadily undergo apoptosis. The objective would be to develop thetechnology to establish cell lines from any number of primary tumorcells, for example primary human leukemia cells, in a reproduciblemanner without altering the native configuration of the signalingpathways in these cells. By introducing nucleic acids encoding peptideswhich inhibit apoptosis, increased cell survival in vitro, and hence theopportunity to study signalling transduction pathways in primary humantumor cells, is accomplished. In addition, these methods may be used forculturing primary cells, i.e. non-tumor cells.

[0131] In a preferred embodiment, the present methods are useful incardiovascular applications. In a preferred embodiment, cardiomyocytesmay be screened for the prevention of cell damage or death in thepresence of normally injurious conditions, including, but not limitedto, the presence of toxic drugs (particularly chemotherapeutic drugs),for example, to prevent heart failure following treatment withadriamycin; anoxia, for example in the setting of coronary arteryocclusion; and autoimmune cellular damage by attack from activatedlymphoid cells (for example as seen in post viral myocarditis andlupus). Candidate bioactive agents are inserted into cardiomyocytes, thecells are subjected to the insult, and bioactive agents are selectedthat prevent any or all of: apoptosis; membrane depolarization (i.e.decrease arrythmogenic potential of insult); cell swelling; or leakageof specific intracellular ions, second messengers and activatingmolecules (for example, arachidonic acid and/or lysophosphatidic acid).

[0132] In a preferred embodiment, the present methods are used to screenfor diminished arrhythmia potential in cardiomyocytes. The screenscomprise the introduction of the candidate nucleic acids encodingcandidate bioactive agents, followed by the application of arrythmogenicinsults, with screening for bioactive agents that block specificdepolarization of cell membrane. This may be detected using patchclamps, or via fluorescence techniques). Similarly, channel activity(for example, potassium and chloride channels) in cardiomyocytes couldbe regulated using the present methods in order to enhance contractilityand prevent or diminish arrhythmias.

[0133] In a preferred embodiment, the present methods are used to screenfor enhanced contractile properties of cardiomyocytes and diminish heartfailure potential. The introduction of the libraries of the inventionfollowed by measuring the rate of change of myosinpolymerization/depolymerization using fluorescent techniques can bedone. Bioactive agents which increase the rate of change of thisphenomenon can result in a greater contractile response of the entiremyocardium, similar to the effect seen with digitalis.

[0134] In a preferred embodiment, the present methods are useful toidentify agents that will regulate the intracellular and sarcolemmalcalcium cycling in cardiomyocytes in order to prevent arrhythmias.Bioactive agents are selected that regulate sodium-calcium exchange,sodium proton pump function, and regulation of calcium-ATPase activity.

[0135] In a preferred embodiment, the present methods are useful toidentify agents that diminish embolic phenomena in arteries andarterioles leading to strokes (and other occlusive events leading tokidney failure and limb ischemia) and angina precipitating a myocardialinfarct are selected. For example, bioactive agents which will diminishthe adhesion of platelets and leukocytes, and thus diminish theocclusion events. Adhesion in this setting can be inhibited by thelibraries of the invention being inserted into endothelial cells(quiescent cells, or activated by cytokines, i.e. IL-1, and growthfactors, i.e. PDGF/EGF) and then screening for peptides that either: 1)downregulate adhesion molecule expression on the surface of theendothelial cells (binding assay); 2) block adhesion molecule activationon the surface of these cells (signaling assay); or 3) release in anautocrine manner peptides that block receptor binding to the cognatereceptor on the adhering cell.

[0136] Embolic phenomena can also be addressed by activating proteolyticenzymes on the cell surfaces of endothelial cells, and thus releasingactive enzyme which can digest blood clots. Thus, delivery of thelibraries of the invention to endothelial cells is done, followed bystandard fluorogenic assays, which will allow monitoring of proteolyticactivity on the cell surface towards a known substrate. Bioactive agentscan then be selected which activate specific enzymes towards specificsubstrates.

[0137] In a preferred embodiment, arterial inflammation in the settingof vasculitis and post-infarction can be regulated by decreasing thechemotactic responses of leukocytes and mononuclear leukocytes. This canbe accomplished by blocking chemotactic receptors and their respondingpathways on these cells. Candidate bioactive libraries can be insertedinto these cells, and the chemotactic response to diverse chemokines(for example, to the IL-8 family of chemokines, RANTES) inhibited incell migration assays.

[0138] In a preferred embodiment, arterial restenosis following coronaryangioplasty can be controlled by regulating the proliferation ofvascular intimal cells and capillary and/or arterial endothelial cells.Candidate bioactive agent libraries can be inserted into these celltypes and their proliferation in response to specific stimuli monitored.One application may be intracellular peptides which block the expressionor function of c-myc and other oncogenes in smooth muscle cells to stoptheir proliferation. A second application may involve the expression oflibraries in vascular smooth muscle cells to selectively induce theirapoptosis. Application of small molecules derived from these peptidesmay require targeted drug delivery; this is available with stents,hydrogel coatings, and infusion-based catheter systems. Peptides whichdownregulate endothelin-1A receptors or which block the release of thepotent vasoconstrictor and vascular smooth muscle cell mitogenendothelin-1 may also be candidates for therapeutics. Peptides can beisolated from these libraries which inhibit growth of these cells, orwhich prevent the adhesion of other cells in the circulation known torelease autocrine growth factors, such as platelets (PDGF) andmononuclear leukocytes.

[0139] The control of capillary and blood vessel growth is an importantgoal in order to promote increased blood flow to ischemic areas(growth), or to cut-off the blood supply (angiogenesis inhibition) oftumors. Candidate bioactive agent libraries can be inserted intocapillary endothelial cells and their growth monitored. Stimuli such aslow oxygen tension and varying degrees of angiogenic factors canregulate the responses, and peptides isolated that produce theappropriate phenotype. Screening for antagonism of vascular endothelialcell growth factor, important in angiogenesis, would also be useful.

[0140] In a preferred embodiment, the present methods are useful inscreening for decreases in atherosclerosis producing mechanisms to findpeptides that regulate LDL and HDL metabolism. Candidate libraries canbe inserted into the appropriate cells (including hepatocytes,mononuclear leukocytes, endothelial cells) and peptides selected whichlead to a decreased release of LDL or diminished synthesis of LDL, orconversely to an increased release of HDL or enhanced synthesis of HDL.Bioactive agents can also be isolated from candidate libraries whichdecrease the production of oxidized LDL, which has been implicated inatherosclerosis and isolated from atherosclerotic lesions. This couldoccur by decreasing its expression, activating reducing systems orenzymes, or blocking the activity or production of enzymes implicated inproduction of oxidized LDL, such as 15-lipoxygenase in macrophages.

[0141] In a preferred embodiment, the present methods are used inscreens to regulate obesity via the control of food intake mechanisms ordiminishing the responses of receptor signaling pathways that regulatemetabolism. Bioactive agents that regulate or inhibit the responses ofneuropeptide Y (NPY), cholecystokinin and galanin receptors, areparticularly desirable. Candidate libraries can be inserted into cellsthat have these receptors cloned into them, and inhibitory peptidesselected that are secreted in an autocrine manner that block thesignaling responses to galanin and NPY. In a similar manner, peptidescan be found that regulate the leptin receptor.

[0142] In a preferred embodiment, the present methods are useful inneurobiology applications. Candidate libraries may be used for screeningfor anti-apoptotics for preservation of neuronal function and preventionof neuronal death. Initial screens would be done in cell culture. Oneapplication would include prevention of neuronal death, by apoptosis, incerebral ischemia resulting from stroke. Apoptosis is known to beblocked by neuronal apoptosis inhibitory protein (NAIP); screens for itsupregulation, or effecting any coupled step could yield peptides whichselectively block neuronal apoptosis. Other applications includeneurodegenerative diseases such as Alzheimer's disease and Huntington'sdisease.

[0143] In a preferred embodiment, the present methods are useful in bonebiology applications. Osteoclasts are known to play a key role in boneremodeling by breaking down “old” bone, so that osteoblasts can lay down“new” bone. In osteoporosis one has an imbalance of this process.Osteoclast overactivity can be regulated by inserting candidatelibraries into these cells, and then looking for bioactive agents thatproduce: 1) a diminished processing of collagen by these cells; 2)decreased pit formation on bone chips; and 3) decreased release ofcalcium from bone fragments.

[0144] The present methods may also be used to screen for agonists ofbone morphogenic proteins, hormone mimetics to stimulate, regulate, orenhance new bone formation (in a manner similar to parathyroid hormoneand calcitonin, for example). These have use in osteoporosis, for poorlyhealing fractures, and to accelerate the rate of healing of newfractures. Furthermore, cell lines of connective tissue origin can betreated with candidate libraries and screened for their growth,proliferation, collagen stimulating activity, and/or prolineincorporating ability on the target osteoblasts. Alternatively,candidate libraries can be expressed directly in osteoblasts orchondrocytes and screened for increased production of collagen or bone.

[0145] In a preferred embodiment, the present methods are useful in skinbiology applications. Keratinocyte responses to a variety of stimuli mayresult in psoriasis, a proliferative change in these cells. Candidatelibraries can be inserted into cells removed from active psoriaticplaques, and bioactive agents isolated which decrease the rate of growthof these cells.

[0146] In a preferred embodiment, the present methods are useful in theregulation or inhibition of keloid formation (i.e. excessive scarring).Candidate libraries inserted into skin connective tissue cells isolatedfrom individuals with this condition, and bioactive agents isolated thatdecrease proliferation, collagen formation, or proline incorporation.Results from this work can be extended to treat the excessive scarringthat also occurs in burn patients. If a common peptide motif is found inthe context of the keloid work, then it can be used widely in a topicalmanner to diminish scarring post burn.

[0147] Similarly, wound healing for diabetic ulcers and other chronic“failure to heal” conditions in the skin and extremities can beregulated by providing additional growth signals to cells which populatethe skin and dermal layers. Growth factor mimetics may in fact be veryuseful for this condition. Candidate libraries can be inserted into skinconnective tissue cells, and bioactive agents isolated which promote thegrowth of these cells under “harsh” conditions, such as low oxygentension, low pH, and the presence of inflammatory mediators.

[0148] Cosmeceutical applications of the present invention include thecontrol of melanin production in skin melanocytes. A naturally occurringpeptide, arbutin, is a tyrosine hydroxylase inhibitor, a key enzyme inthe synthesis of melanin. Candidate libraries can be inserted intomelanocytes and known stimuli that increase the synthesis of melaninapplied to the cells. Bioactive agents can be isolated that inhibit thesynthesis of melanin under these conditions.

[0149] In a preferred embodiment, the present methods are useful inendocrinology applications. The retroviral peptide library technologycan be applied broadly to any endocrine, growth factor, cytokine orchemokine network which involves a signaling peptide or protein thatacts in either an endocrine, paracrine or autocrine manner that binds ordimerizes a receptor and activates a signaling cascade that results in aknown phenotypic or functional outcome. The methods are applied so as toisolate a peptide which either mimics the desired hormone (i.e.,insulin, leptin, calcitonin, PDGF, EGF, EPO, GMCSF, IL1-17, mimetics) orinhibits its action by either blocking the release of the hormone,blocking its binding to a specific receptor or carrier protein (forexample, CRF binding protein), or inhibiting the intracellular responsesof the specific target cells to that hormone. Selection of peptideswhich increase the expression or release of hormones from the cellswhich normally produce them could have broad applications to conditionsof hormonal deficiency.

[0150] In a preferred embodiment, the present methods are useful ininfectious disease applications. Viral latency (herpes viruses such asCMV, EBV, HBV, and other viruses such as HIV) and their reactivation area significant problem, particularly in immunosuppressed patients(patients with AIDS and transplant patients). The ability to block thereactivation and spread of these viruses is an important goal. Celllines known to harbor or be susceptible to latent viral infection can beinfected with the specific virus, and then stimuli applied to thesecells which have been shown to lead to reactivation and viralreplication. This can be followed by measuring viral titers in themedium and scoring cells for phenotypic changes. Candidate libraries canthen be inserted into these cells under the above conditions, andpeptides isolated which block or diminish the growth and/or release ofthe virus. As with chemotherapeutics, these experiments can also be donewith drugs which are only partially effective towards this outcome, andbioactive agents isolated which enhance the virucidal effect of thesedrugs.

[0151] One example of many is the ability to block HIV-1 infection.HIV-1 requires CD4 and a co-receptor which can be one of several seventransmembrane G-protein coupled receptors. In the case of the infectionof macrophages, CCR-5 is the required co-receptor, and there is strongevidence that a block on CCR-5 will result in resistance to HIV-1infection. There are two lines of evidence for this statement. First, itis known that the natural ligands for CCR-5, the CC chemokines RANTES,MIP1a and MIP1b are responsible for CD8+ mediated resistance to HIV.Second, individuals homozygous for a mutant allele of CCR-5 arecompletely resistant to HIV infection. Thus, an inhibitor of theCCR-5/HIV interaction would be of enormous interest to both biologistsand clinicians. The extracellular anchored constructs offer superb toolsfor such a discovery. Into the transmembrane, epitope tagged,glycine-serine tethered constructs (ssTM V G20 E TM), one can place arandom, cyclized peptide library of the general sequence CXXXXXXXXXXC orC-(X)_(n)-C (SEQ ID NO:50). Then one infects a cell line that expressesCCR-5 with retroviruses containing this library. Using an antibody toCCR-5 one can use FACS to sort desired cells based on the binding ofthis antibody to the receptor. All cells which do not bind the antibodywill be assumed contain inhibitors of this antibody binding site. Theseinhibitors, in the retroviral construct can be further assayed for theirability to inhibit HIV-1 entry.

[0152] Viruses are known to enter cells using specific receptors to bindto cells (for example, HIV uses CD4, coronavirus uses CD13, murineleukemia virus uses transport protein, and measles virus usesCD44) andto fuse with cells (HIV uses chemokine receptor). Candidate librariescan be inserted into target cells known to be permissive to theseviruses, and bioactive agents isolated which block the ability of theseviruses to bind and fuse with specific target cells.

[0153] In a preferred embodiment, the present invention finds use withinfectious organisms. Intracellular organisms such as mycobacteria,listeria, salmonella, pneumocystis, yersinia, leishmania, T. cruzi, canpersist and replicate within cells, and become active inimmunosuppressed patients. There are currently drugs on the market andin development which are either only partially effective or ineffectiveagainst these organisms. Candidate libraries can be inserted intospecific cells infected with these organisms (pre- or post-infection),and bioactive agents selected which promote the intracellulardestruction of these organisms in a manner analogous to intracellular“antibiotic peptides” similar to magainins. In addition peptides can beselected which enhance the cidal properties of drugs already underinvestigation which have insufficient potency by themselves, but whencombined with a specific peptide from a candidate library, aredramatically more potent through a synergistic mechanism. Finally,bioactive agents can be isolated which alter the metabolism of theseintracellular organisms, in such a way as to terminate theirintracellular life cycle by inhibiting a key organismal event.

[0154] Antibiotic drugs that are widely used have certain dosedependent, tissue specific toxicities. For example renal toxicity isseen with the use of gentamicin, tobramycin, and amphotericin;hepatotoxicity is seen with the use of INH and rifampin; bone marrowtoxicity is seen with chloramphenicol; and platelet toxicity is seenwith ticarcillin, etc. These toxicities limit their use. Candidatelibraries can be introduced into the specific cell types where specificchanges leading to cellular damage or apoptosis by the antibiotics areproduced, and bioactive agents can be isolated that confer protection,when these cells are treated with these specific antibiotics.

[0155] Furthermore, the present invention finds use in screening forbioactive agents that block antibiotic transport mechanisms. The rapidsecretion from the blood stream of certain antibiotics limits theirusefulness. For example penicillins are rapidly secreted by certaintransport mechanisms in the kidney and choroid plexus in the brain.Probenecid is known to block this transport and increase serum andtissue levels. Candidate agents can be inserted into specific cellsderived from kidney cells and cells of the choroid plexus known to haveactive transport mechanisms for antibiotics. Bioactive agents can thenbe isolated which block the active transport of specific antibiotics andthus extend the serum halflife of these drugs.

[0156] In a preferred embodiment, the present methods are useful in drugtoxicities and drug resistance applications. Drug toxicity is asignificant clinical problem. This may manifest itself as specifictissue or cell damage with the result that the drug's effectiveness islimited. Examples include myeloablation in high dose cancerchemotherapy, damage to epithelial cells lining the airway and gut, andhair loss. Specific examples include adriamycin induced cardiomyocytedeath, cisplatinin-induced kidney toxicity, vincristine-induced gutmotility disorders, and cyclosporin-induced kidney damage. Candidatelibraries can be introduced into specific cell types with characteristicdrug-induced phenotypic or functional responses, in the presence of thedrugs, and agents isolated which reverse or protect the specific celltype against the toxic changes when exposed to the drug. These effectsmay manifest as blocking the drug induced apoptosis of the cell ofinterest, thus initial screens will be for survival of the cells in thepresence of high levels of drugs or combinations of drugs used incombination chemotherapy.

[0157] Drug toxicity may be due to a specific metabolite produced in theliver or kidney which is highly toxic to specific cells, or due to druginteractions in the liver which block or enhance the metabolism of anadministered drug. Candidate libraries can be introduced into liver orkidney cells following the exposure of these cells to the drug known toproduce the toxic metabolite. Bioactive agents can be isolated whichalter how the liver or kidney cells metabolize the drug, and specificagents identified which prevent the generation of a specific toxicmetabolite. The generation of the metabolite can be followed by massspectrometry, and phenotypic changes can be assessed by microscopy. Sucha screen can also be done in cultured hepatocytes, cocultured withreadout cells which are specifically sensitive to the toxic metabolite.Applications include reversible (to limit toxicity) inhibitors ofenzymes involved in drug metabolism.

[0158] Multiple drug resistance, and hence tumor cell selection,outgrowth, and relapse, leads to morbidity and mortality in cancerpatients. Candidate libraries can be introduced into tumor cell lines(primary and cultured) that have demonstrated specific or multiple drugresistance. Bioactive agents can then be identified which confer drugsensitivity when the cells are exposed to the drug of interest, or todrugs used in combination chemotherapy. The readout can be the onset ofapoptosis in these cells, membrane permeability changes, the release ofintracellular ions and fluorescent markers. The cells in which multidrugresistance involves membrane transporters can be preloaded withfluorescent transporter substrates, and selection carried out forpeptides which block the normal efflux of fluorescent drug from thesecells. Candidate libraries are particularly suited to screening forpeptides which reverse poorly characterized or recently discoveredintracellular mechanisms of resistance or mechanisms for which few or nochemosensitizers currently exist, such as mechanisms involving LRP (lungresistance protein). This protein has been implicated in multidrugresistance in ovarian carcinoma, metastatic malignant melanoma, andacute myeloid leukemia. Particularly interesting examples includescreening for agents which reverse more than one important resistancemechanism in a single cell, which occurs in a subset of the most drugresistant cells, which are also important targets. Applications wouldinclude screening for peptide inhibitors of both MRP (multidrugresistance related protein) and LRP for treatment of resistant cells inmetastatic melanoma, for inhibitors of both p-glycoprotein and LRP inacute myeloid leukemia, and for inhibition (by any mechanism) of allthree proteins for treating pan-resistant cells.

[0159] In a preferred embodiment, the present methods are useful inimproving the performance of existing or developmental drugs. First passmetabolism of orally administered drugs limits their oralbioavailability, and can result in diminished efficacy as well as theneed to administer more drug for a desired effect. Reversible inhibitorsof enzymes involved in first pass metabolism may thus be a usefuladjunct enhancing the efficacy of these drugs. First pass metabolismoccurs in the liver, thus inhibitors of the corresponding catabolicenzymes may enhance the effect of the cognate drugs. Reversibleinhibitors would be delivered at the same time as, or slightly before,the drug of interest. Screening of candidate libraries in hepatocytesfor inhibitors (by any mechanism, such as protein downregulation as wellas a direct inhibition of activity) of particularly problematicalisozymes would be of interest. These include the CYP3A4 isozymes ofcytochrome P450, which are involved in the first pass metabolism of theanti-HIV drugs saquinavir and indinavir. Other applications couldinclude reversible inhibitors of UDP-glucuronyltransferases,sulfotransferases, N-acetyltransferases, epoxide hydrolases, andglutathione S-transferases, depending on the drug. Screens would be donein cultured hepatocytes or liver microsomes, and could involveantibodies recognizing the specific modification performed in the liver,or cocultured readout cells, If the metabolite had a differentbioactivity than the untransformed drug. The enzymes modifying the drugwould not necessarily have to be known, if screening was for lack ofalteration of the drug.

[0160] In a preferred embodiment, the present methods are useful inimmunobiology, inflammation, and allergic response applications.Selective regulation of T lymphocyte responses is a desired goal inorder to modulate immune-mediated diseases in a specific manner.Candidate libraries can be introduced into specific T cell subsets (TH1,TH2, CD4+, CD8+, and others) and the responses which characterize thosesubsets (cytokine generation, cytotoxicity, proliferation in response toantigen being presented by a mononuclear leukocyte, and others) modifiedby members of the library. Agents can be selected which increase ordiminish the known T cell subset physiologic response. This approachwill be useful in any number of conditions, including: 1) autoimmunediseases where one wants to induce a tolerant state (select a peptidethat inhibits T cell subset from recognizing a self-antigen bearingcell); 2) allergic diseases where one wants to decrease the stimulationof IgE producing cells (select peptide which blocks release from T cellsubsets of specific B-cell stimulating cytokines which induce switch toIgE production); 3) in transplant patients where one wants to induceselective immunosuppression (select peptide that diminishesproliferative responses of host T cells to foreign antigens); 4) inlymphoproliferative states where one wants to inhibit the growth orsensitize a specific T cell tumor to chemotherapy and/or radiation; 5)in tumor surveillance where one wants to inhibit the killing ofcytotoxic T cells by Fas ligand bearing tumor cells; and 5) in T cellmediated inflammatory diseases such as Rheumatoid arthritis, Connectivetissue diseases (SLE), Multiple sclerosis, and inflammatory boweldisease, where one wants to inhibit the proliferation of disease-causingT cells (promote their selective apoptosis) and the resulting selectivedestruction of target tissues (cartilage, connective tissue,oligodendrocytes, gut endothelial cells, respectively).

[0161] Regulation of B cell responses will permit a more selectivemodulation of the type and amount of immunoglobulin made and secreted byspecific B cell subsets. Candidate libraries can be inserted into Bcells and bioactive agents selected which inhibit the release andsynthesis of a specific immunoglobulin. This may be useful in autoimmunediseases characterized by the overproduction of auto antibodies and theproduction of allergy causing antibodies, such as IgE. Agents can alsobe identified which inhibit or enhance the binding of a specificimmunoglobulin subclass to a specific antigen either foreign of self.Finally, agents can be selected which inhibit the binding of a specificimmunoglobulin subclass to its receptor on specific cell types.Similarly, agents which affect cytokine production may be selected,generally using two cell systems. For example, cytokine production frommacrophages, monocytes, etc. may be evaluated. Similarly, agents whichmimic cytokines, for example erythropoetin and IL1-17, may be selected,or agents that bind cytokines such as TNF-α, before they bind theirreceptor.

[0162] Antigen processing by mononuclear leukocytes (ML) is an importantearly step in the immune system's ability to recognize and eliminateforeign proteins. Candidate agents can be inserted into ML cell linesand agents selected which alter the intracellular processing of foreignpeptdes and sequence of the foreign peptide that is presented to T cellsby MLs on their cell surface in the context of Class II MHC. One canlook for members of the library that enhance immune responses of aparticular T cell subset (for example, the peptide would in fact work asa vaccine), or look for a library member that binds more tightly to MHC,thus displacing naturally occurring peptides, but nonetheless the agentwould be less immunogenic (less stimulatory to a specific T cell clone).This agent would in fact induce immune tolerance and/or diminish immuneresponses to foreign proteins. This approach could be used intransplantation, autoimmune diseases, and allergic diseases.

[0163] The release of inflammatory mediators (cytokines, leukotrienes,prostaglandins, platelet activating factor, histamine, neuropeptides,and other peptide and lipid mediators) is a key element in maintainingand amplifying aberrant immune responses. Candidate libraries can beinserted into MLs, mast cells, eosinophils, and other cellsparticipating in a specific inflammatory response, and bioactive agentsselected which inhibit the synthesis, release and binding to the cognatereceptor of each of these types of mediators.

[0164] In a preferred embodiment, the present methods are useful inbiotechnology applications. Candidate library expression in mammaliancells can also be considered for other pharmaceutical-relatedapplications, such as modification of protein expression, proteinfolding, or protein secretion. One such example would be in commercialproduction of protein pharmaceuticals in CHO or other cells. Candidatelibraries resulting in bioactive agents which select for an increasedcell growth rate (perhaps peptides mimicking growth factors or acting asagonists of growth factor signal transduction pathways), for pathogenresistance (see previous section), for lack of sialylation orglycosylation (by blocking glycotransferases or rerouting trafficking ofthe protein in the cell), for allowing growth on autoclaved media, orfor growth in serum free media, would all increase productivity anddecrease costs in the production of protein pharmaceuticals.

[0165] Random peptides displayed on the surface of circulating cells canbe used as tools to identify organ, tissue, and cell specific peptidetargeting sequences. Any cell introduced into the bloodstream of ananimal expressing a library targeted to the cell surface can be selectedfor specific organ and tissue targeting. The bioactive agent sequenceidentified can then be coupled to an antibody, enzyme, drug, imagingagent or substance for which organ targeting is desired.

[0166] Other agents which may be selected using the present inventioninclude: 1) agents which block the activity of transcription factors,using cell lines with reporter genes; 2) agents which block theinteraction of two known proteins in cells, using the absence of normalcellular functions, the mammalian two hybrid system or fluorescenceresonance energy transfer mechanisms for detection; and 3) agents may beidentified by tethering a random peptide to a protein binding region toallow interactions with molecules sterically close, i.e. within asignalling pathway, to localize the effects to a functional area ofinterest.

[0167] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference intheir entirety.

EXAMPLES Example 1 Proof of Concept Experiments

[0168] A number of systems were used to prove that the retroviralconstructs outlined herein were able to result in a selectablephenotype.

[0169] Bcl2 and CPP32 Protection from apoptosis

[0170] It is known that Bcl2 and the CPP32 peptide is able to inhibitapoptosis induced by tumor necrosis factor and cycloheximide.

[0171] Apotag assay: TUNEL (TdT-mediated dUTP-fluorescein nick endlabeling) Boehringer Mannheim kit, catalog no. 168795

[0172] 3T3 cells transiently infected with either MFGLacZ, BCL2, orCPP322 plasmids were grown to 50% confluence at the time of inductionwith hTNFa (50 ng/ml media) and cycloheximide (100 mg/ml media) for 6hours. Cells were washed at 6 hours and harvested at 24 hours afterinduction. Cells were harvested by pooling all media from cells (inorder to collect any apoptotic, floating cells) with the washings andtrypsinized cells. The cells were spun and washed with PBS containing 1%BSA, transferred to an eppendorf tube and the wash repeated once. Cellswere fixed in 4% paraformaldehyde at room temperature for 30 minutes,washed in PBS/BSA, then resuspended in permeabilisation buffer for 2minutes on ice. After permeabilisation, cells were washed twice inPBS/BSA and incubated at 37° C. for 1 hour with labeling buffercontaining fluoresceinated dUTP, unlabeled nucleotide mixture andterminal deoxynucleotidyl transferase (TdT). Cells were washed twicewith PBS/BSA, resuspended in PBS/BSA and transferred to a FACS tube foranalysis. Samples were also visualized under the fluorescencemicroscope. The results showed that expression of Bcl2 or the CPP32peptide in 3T3 cells from an MSCV retroviral promoter in vivo was ableto inhibit apoptosis induced by tumor necrosis factor and cycloheximide.

[0173] Propidium Iodide staining of fixed cells to assay for apoptosis:(Sherwood and Schimke, Methods in Cell Biology, 46:77-87, 1995) 3T3cells transiently infected with MFGLacZ, Bcl2, or CPP32 were plated andtreated with TNF/CXH as described above, and harvested and washed asabove. Cells were then resuspended in 70% ethanol in PBS at 4° C. andkept at 4° C. overnight. When ready to FACS, cells were stained withpropidium iodide as follows. Cells were spun at 14,000 RPM for 10seconds and washed once with PBS/BSA. Cells were then resuspended in 50ml staining solution (PBS with 50 mg/ml RNase A (DNase-free) with 10mg/ml propidium iodide) and incubated at 37° C for at least 1 hour.Cells were then pelleted and resuspended in PBS/BSA solution containing10 mg/ml propidium iodide and analyzed by FACS scanning. The resultsshowed that expression of BCL2 or CPP32 peptide in 3T3 cells was able toinhibit apoptosis induced by tumor necrosis factor and cycloheximide asmeasure by Pl staining of cells, extending our previous results.

[0174] Ethidium Bromide/Acridine Orange Staining of BAF3 Cells to StudvCell Morphology: BAF3 cells were infected with WZL IRES NEO retroviralvectors containing no insert (WIN) or DNA coding for LacZ (ZIN), Bcl2(BIN), CPP32 peptide (CIN), or scrambled peptide control (PIN). Cellswere selected in G418 after infection with above retroviral vectors andsurvivors were stimulated with 5 mg/ml FAS antibody. After stimulation,cells were stained with ethidium bromide and acridine orange (2 mg/mleach) and visualized under the fluorescence microscope using theultraviolet filter. 250 cells were counted and the percent of cellswhich were apoptotic were calculated. Similar to results obtained in 3T3cells stimulated with TNF/CXH, the CPP32 encoding vectors are able toinhibit FAS induced apoptosis. The peptide control also had an effect inthis system approximately half of that seen with BCL2 or the CPP32peptide.

[0175] Enzymatic Assay of CPP32 activity: CPP32 Assay Kit: ClontechCPP32 Colorimetric Assay Kit (Cat. No. K2027-2): 3T3 cells were infectedwith the vectors described in Part I, section C, and selected in G418media prior to assay. 6-well plates of 3T3 cells at near confluence werestimulated with TNF/CXH as described above and harvested at 30 min, 1, 2and 4 hours after stimulation as follows. Cells were trypsinized andcollected as described above. After transfering to an eppendorf tube,cells were spun and resuspended in 50 ml chilled Cell Lysis Buffer.Cells were incubated for 10 minutes on ice, then 50 ml of 2× ReactionBuffer containing DTT was added to each tube. 5 ml of the calorimetricconjugated substrate (DEVD-paranitroanilide, 50 mM final concentration)was added to each tube and incubated at 37° C. for 30 minutes. Sampleswere transferred to a 96 well plate and read on a spectrophotometer atO.D. of 405. The results showed that cell extracts from WIN cells haveincreased CPP32 enzyme activity at 2 hours as measured by cleavage ofDEVD-pNA substrate to its colorometrically detectable form pNA. By 4hours, cells have begun to die and the activity is decreased. In cellscontaining BCL2 or the CPP32 peptide inhibitor, this rise in activity isnot seen. In the case of BCL2, it should be due to inhibition ofapoptosis upstream of the enzyme. With CPP32 inhibitor peptide, itshould be due to direct inhibition of enzymatic activity. These in vitroresults are consistent with the results seen in cell death assaysdescribed above.

[0176] Localization Studies Using PKC Inhibitor

[0177] Murine 10T1/2 Clone 8 cells were stimulated with PMA which isknown to cause translocation of PKC from the cytoplasm to the nucleus.This translocation is thought to be mediated through binding to aprotein at the site of action, termed a RAC (receptor for activatedprotein kinase C) protein. Uninfected clone 8 cells were compared tocells infected with pBabe puro retroviral constructs containingsequences coding for either Flu-epitope (MGGGYPYDVPDYAGSLZ) (SEQ IDNO:51) tagged scrambled peptide control or inhibitor peptide(GKQKTKTIKGPP) (SEQ ID NO:52) which is identical to the C2 region of allthe PKC isozymes. We then assayed the cells by immunohistochemistryusing an antibody specific for PKCa and visualized with a secondaryantibody conjugated to horseradish peroxidase. This experiment was doneat two different cell densities as follows:

[0178] 1. Cells were plated at 2,000 cells/cm² onto 22 mm squarepolylysine coated coverslips and allowed to grow for 2 days. On 3/20,cells were nearly confluent. Cells were replated at a lower density andassayed with identical conditions on 3/27.

[0179] 2. PMA was added at 10⁻⁵M to the media for 30 minutes at 37° C.

[0180] 3. Cells were rinsed with SCB buffer (physiologic bufferprewarmed to 37° C. before use) and then placed into 3.7% glutaraldehydein SCB buffer for 20 minutes at 37° C.

[0181] 4.Cells were then washed in SCB buffer then incubated with SCBT(SCB containing 0.1% Triton X-100) for 10 minutes at room temperature.

[0182] 5.Coverslips were removed from the 6-well plate and dip washed in0.1% tween/PBS at room temperature and placed onto parafilm in a coveredcontainer.

[0183] 6.Coverslips were incubated with 1.5% goat blocking serum in PBSwith agitation in a humidified environment at room temperature.

[0184] 7.Solutions were aspirated off the coverslip and coverslips werethen washed with PBS. Primary anti-PKCa antibody was placed ontocoverslips and incubated for 30 minutes at room temperature as above. A1:500 dilution of antibody was used in all experiments.

[0185] 8.Coverslips were then washed with PBS three times and thenincubated for 30 minutes at room temperature with biotin-conjugatedsecond step antibody as provided in Santa Cruz ABC ImmunoStain Sytemskit. Coverslips were then washed three times with PBS.

[0186] 9. Coverslips were then incubated in avidin biotin enzyme reagent(as supplied with kit) for 30 minutes at room temperature. Coverslipswere then washed for 10 minutes in PBS after being placed back into6-well plates.

[0187] 10. Coverslips were rinsed with 0.5% Triton X-100/PBS for 30seconds and incubated in DAB solution for 5 minutes. Reaction wasstopped by addition of distilled water to well.

[0188] 11. Coverslips were then dehydrated through alcohols and xyleneand mounted onto slides with Permount and visualised and photographed bylight microscopy. The result showed that basically, control clone 8cells showed predominantly cytoplasmic and perinuclear staining, whilePMA induced cells consistently showed translocation to the nucleus.Cells infected with constructs coding for the scrambled peptide showedsimilar staining. Cells infected with constructs coding for peptidesidentical to the C2 region of PKC showed predominantly cytoplasmic andperinuclear staining in both control and PMA induced cells suggestingthat this peptide is able to specifically inhibit translocation ofactivated PKCa to its RAC protein upon stimulation of the cells withPMA. It is also possible using similarly infected cells to see thedownstream results of peptide expression upon gene activity. Cells wereinfected with retroviruses expressing either the PKCb2.1, PKC2.1peptide, a dominant negative ras protein control, combinations of theseviruses, or no virus at all. Cells were stimulated with PMA at 100ng/ml, PDGF-AA, or PDGF-BB. mRNA was prepared and northern blots wereperformed for fos gene expression (induced by PKC activation) or theribosomal protein P0, a loading control mRNA whose expression is notknown to be acted upon by signaling systems induced by PKC. The PKCpeptides can markedly reduce expression of the fos gene mRNA. Indeed, anunexpected result was that under certain conditions there is activationof the mRNA expression. This latter results confirms that novel outcomescan occur upon expression of peptides within cells.

Example 2 pBabe Puro Retroviral Libraries and Apoptosis

[0189] A series of retroviral constructs have been designed forexpression of randomized and biased peptides within target cellpopulations. The peptide is expressed from a retroviral promoter. Thetranslation unit has several important components. Glycine following theinitiator methionine at the amino terminus stabilizes the peptide andenhances cytoplasmic half-life, according to Varshavsky's N-End Rule. Insome constructs, a nine amino acid flu epitope tag has been incorporatedto permit co-precipitation of the rare peptide and any molecule to whichit has affinity, by using monoclonal antibodies to the epitope. Glycinesare encoded before and after the random/biased expression productencoding regions to provide some molecular flexibility. Twocarboxyl-terminal prolines are encoded to confer stability tocarboxypeptidase.

[0190] For construction of a large library two primers were made(schematized in FIG. 1). The first, designated the random peptideprimer, consists of 1 ) a complementary region for vector priming, 2)the regions mentioned above, and 3) a random or biased expressionproduct region, were presented as a 30 base sequence encoding a peptideof length 10 amino acids. In addition, we have inserted a stop codon inall three reading frames in case of minor deletions or insertions in therandom region. The design of the primer ensures a glycine/prolinetermination in most reading frames. The second primer is downstream inthe vector and primes a region of the plasmid that contains a unique NotI site. These primers are used to create a library of fragments, eachcontaining a different nucleotide sequence that each potentially encodesa different peptide. These families of fragments are ligated to vectorfragments containing puromycin selection sequence, a 3′LTR, and abacterial origin of replication. The ligation products are thenelectroporated into E. cold and DNA is prepared from the resultinglibrary. Using this technique, we have constructed independent randomlibraries with up to 2×10⁸ unique inserts. Sequencing multipleindividual inserts demonstrates they have the structure as defined byPrimer 1, and the peptides encoded are random. Such libraries thus madecontain subsets of the total 10¹³ predicted peptides.

[0191] Generation of Retroviral Peptide Libraries

[0192] A scheme for generating a peptide library in the pBabe Purovector is shown in FIG. 2. Primers for PCR were synthesized, purifiedand deprotected according to standard protocols. Primer 1, complementaryto polylinker sequences in the pBabe Puro retroviral construct, has thesequence 5′ GCT TAG CAA GAT CTC TAC GGT GGA CCK NNK NNK NNK NNK NNK NNKNNK NNK NNK NNC CCC ACT CCC ATG GTC CTA CGT ACC ACC ACA CTG GG 3′ (SEQID NO:53). N represents any of the four bases; K is limited to G or T.Primer 2 has the sequence 5′ GCT TAG CAA GAT CTG TGT GTC AGT TAG GGT GTGG 3′ (SEQ ID NO:54) and is complementary to sequences within the pUC18origin of replication. PCR was carried out for 8 rounds using primer 1,primer 2, Babe Puro as template, and a mixture of Taq DNA Polymerase(Promega) and Deep Vent DNA Polymerase (New England Biolabs) in a ratioof 128 Taq: 1 Deep Vent as described in Barnes (1994) Proc. Natl. Acad.Sci. USA, 91, pp. 2216-2220. The amplified PCR product was purified,digested with restriction enzymes Bgl II and Not I (Promega), purifiedagain and ligated with the corresponding Bam HI-Not I fragment of pBabePuro. After transformation the resulting library contained ˜2×10⁸clones, greater than 80% of which contained inserts.

[0193] pMSCV-PC and pBabeMN-PC Retroviral Construct Libraries

[0194] Oligonucleotides were synthesized and purified according tostandard protocols. The “library” oligonucleotides have the sequence 5′CTG GAG MC CAG GAC CAT GGG C (NNK)10 GGG CCC CCT TM ACC ATT AAA T 3′(SEQ ID NO:55) or 5′ CTG GAG AAC CAG GAC CAT GGG CNN KNN KNN KCC TCC CNNKCC TNN KNN KGG GCC CCC TTA AAC CAT TAA AT 3′ (SEQ ID NO:56). A thirdoligonucleotide (“constant”), complementary to the 3′ ends of thelibrary oligonucleotides, has the sequence 5′TCA TGC ATC CM TTT AAT GGTTTA AG 3′ (SEQ ID NO:57). As shown in FIG. 3, each libraryoligonucleotide is annealed to the constant oligonucleotide, convertedto double stranded DNA with Sequenase (United States Biochemical) orKlenow (Promega), digested with restriction enzyme Bst XI (New EnglandBiolabs), and purified and ligated with the appropriate Bst XI-digestedretroviral construct. Transformation efficiencies are ˜2×10⁸ clones permicrogram of ligated DNA, greater than 90% of which contain an insert. Arepresentative retrovirus is shown in FIG. 4; see also, retroviralnucleotide sequence below: Retroviral vector with presentationconstruct. TGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAAGGCCATTTTGCAA (SEQID NO:58) GGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTATTCCCAATAAAGCCTCTTGGTGTTTGCATCCGAATCGTGGACTGGCTGATCCTTGGGAGGGTCTCCTCAGATTGATTGACTGCCCACCTCGGGGGTCTTTCATTTGGAGGTTCCAGCGAGATTTGGAGACCCCTGCCTAGGGACCACCGACCCCCCCGCCGGGAGGTAAGCTGGCCAGCGGTCGTTTCCTGTCTGTCTCTGTCTTTGTGCGTGTTTGTGCCGGCATCTAATGTTTGCGCCTGCGTCTGTACTAGTTAGCTAACTAGCTCTGTATCTGGCGGACCCGTGGTGGAACTGACGAGTTCTGAACACCCGGCCGCAACCCTGGGAGACGTCCCAGGGACTTTGGGGGCCGTTTTTGTGGCCCGACCTGAGGAAGGGAGTCGATGTGGAATCCGACCCCGTCAGGATATGTGGTTCTGGTAGGAGACGAGAACCTAAAACAGTTCCCGCCTCCGTCTGAATTTTTGCTTTCGGTTTGGAACCGAAGCCGCGCGTCTTGTCTGCTGCAGCGCTGCAGCATCGTTCTGTGTTCTCTCTGTCTGACTGTGTTTCTGTATTTGTCTGAAAATTAGGGCCAGACTGTTACCACTCCCTTAAGTTTGACCTTAGGTCACTGGAAAGATGTCGAGCGGATCGCTCACAACCAGTCGGTAGATGTCAAGAAGAGACGTTGGGTTACCTTCTGCTCTGCAGAATGGCCAACCTTTAACGTCGGATGGCCGCGAGACGGCACCTTTAACCGAGACCTCATCACCCAGGTTAAGATCAAGGTCTTTTCACCTGGCCCGCATGGACACCCAGACCAGGTCCCCTACATCGTGACCTGGGAAGCCTTGGCTTTTGACCCCCCTCCCTGGGTCAAGCCCTTTGTACACCCTAAGCCTCCGCCTCCTCTTCCTCCATCCGCCCCGTCTCTCCCCCTTGAACCTCCTCGTTCGACCCCGCCTCGATCCTCCCTTTATCCAGCCCTCACTCCTTCTCTAGGCGCCGGAATTCCAGGACCATGGGCGGGCCCCCTTAAACCATTAAATTGGTAAAATAAAGGATCCGTCGACCTGCAGCCAAGCTTATCGATAAAATAAAAGATTTTATTTAGTCTCCAGAAAAAGGGGGGAATGAAAGACCCCACCTGTAGGTTTGGCAAGCTAGCTTAAGTAACGCCATTTTGCAAGGCATGGAAAATACATAACTGAGAATAGAGAAGTTCAGATCAAGGTTAGGAACAGAGAGACAGCAGAATATGGGCCAAACAGGATATCTGTGGTAAGCAGTTCCTGCCCCGGCTCAGGGCCAAGAACAGATGGTCCCCAGATGCGGTCCCGCCCTCAGCAGTTTCTAGAGAACCATCAGATGTTTCCAGGGTGCCCCAAGGACCTGAAAATGACCCTGTGCCTTATTTGAACTAACCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATAGACTGCGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCATTCGTAATCATGGTCATAGCTGTTTCCTGTGTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACGCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGGGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCGGGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAAAATAGGCGTATCACGAGGCCCTTTCGTCTCGCGCGTTTCGGTGATGACGGTGAAAACCTCTGACACATGCAGCTCCCGGAGACGGTCACAGCTTGTCTGTAAGCGGATGCCGGGAGCAGACAAGCCCGTCAGGGCGCGTCAGCGGGTGTTGGCGGGTGTCGGGGCTGGCTTAACTATGCGGCATCAGAGCAGATTGTACTGAGAGTGCACCATATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGCGCCATTCGCCATTCAGGCTGCGCAACTGTTGGGAAGGGGGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGAGGGCCAGTGCCAGGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGGACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCGACGCCGAAACAA~3CGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAG

[0195] Peptide Library Infection of a Factor-dependent Line andOutgrowth of an Apoptosis-Resistant Line

[0196] The Baf/3 cell line is an IL-3 dependent cell that undergoesrapid apoptosis in the absence af IL-3. Thus it makes an attractive cellline for dominant effector peptides. Cells expressing a peptide thatinhibits apoptosis are readily selected against the background of dyingcells. We chose this cell line as a model for demonstrating peptideselection.

[0197] A retroviral library containing 5×10⁶ independent peptide insertswas transfected into BOSC23 cells and converted into retrovirus with anappropriate titer of 5×10⁵ per ml. Twelve ml of viral supernatant wasused to infect 6×10⁶ Baf/3cells (2 ml [per infection of 1×10⁶ cells inindependent infections). Cells were grown for 3 days after infection inthe presence of IL-3 to allow retroviral integration and peptideexpression. After three days IL-3 was withdrawn and the cells allowed togrow for two weeks. After two weeks, one well of six had outgrowth ofcells that survive in the absence of IL-3, indicating the presence of anapoptosis-inhibiting peptide. Peptides derived in this manner may effectthe IL-3 independence by positive dominancy (i.e., mimic or circumventthe positive regulatory role of IL-3) or by inhibition (i.e., preventthe apoptosis process upon IL-3 withdrawal).

Example 3 pMSCVpc Vector Construction and Apoptosis

[0198] The retroviral vector pMSCVpc was prepared by cloning an insertcontaining sequences encoding a Kozak translation initiation sequence,BstXI sites for cloning library inserts, NruI and XhoI sites and stopcodons in all three reading frames, into the EcoRI and BamHI sites ofpMSCV neo.

[0199] BstX I Restriction Digestion

[0200] 200 μg pMSCVpc vector DNA was combined with 40 μl 10×NEBuffer 3and 30 μl BstX I (10 units/μl) in a total volume of 400 μl. The samplewas incubated overnight at 55° C., phenol extracted, and digested withXhoI, and purified on a potassium acetate step gradient using 10, 15, 20and 25% solutions of potassium acetate. The DNA was PreciPitated, with arecovery of 40%.

[0201] Library Insert Preparation

[0202] Oligonucleotide Synthesis

[0203] Oligonucleotides (OL) with the following sequences weresynthesized: OL-1: 5′-CTG GAG AAC CAG GAC CAT GGG CAA GAG AAA GGG CGATGA GGT GGA TGG AGT GGG GCC CCC TTA AAC CAT TAA AT 3′ (SEQ ID NO:59).The underlined region encodes a peptide with the sequenceMGKRKGDEVDGVGPP (SEQ ID NO:60). This peptide was shown to inhibitFas-mediated and Staurosporin induced apoptosis when expressed in cellswith a retrovirus. OL-2: 5′-CTG GAG MC CAG GAC CAT GGG CM GAG MA GGG CNNKNN KNN KGA KNN KGT GGG GCC CCC TTA MC CAT TM AT-3′ (SEQ ID NO:61)Variable region: N=A, C, G, T (equimolar) K=G, T (equimolar) Limitingthe K position of each codon to G or T reduces stop codon generation andcodon usage bias. The underlined region encodes a randomized peptidewith the sequence MGKRKGXXXD/EXVGPPA (SEQ ID NO:62).

[0204] OL-3: 5′-TCA TGC ATC CAA TTT AAT GGT TTA AG-3′ (SEQ ID NO:63) The15 3′-bases of OL-3 are complementary to the 15 3′-bases of OL-1 andOL-2.

[0205] OL-1 and OL-2 were synthesized at 1 μM scale, while OL-3 wassynthesized at standard 40 nM scale. All of the oligos were synthesizedwith trityl-on, deprotected and purified on OPC columns according to themanufacturer's directions (Applied Biosystems). Each oligo wasresuspended in 200 μl 10 mM Tris pH 8.5 without EDTA. The DNAconcentration was determined by measuring the absorbance at 260 nm.

[0206] PCR was done with 50 pmole of either OL-1 or OL-2 and 50 pmole ofOL-3. Phenol extraction and ethanol precipitation was done, and theresulting DNA was run on a 10% nondenaturing 10% acrylamide gel, withethidium bromide staining.

[0207] The samples were quantitated, ligated, precipitated andelectroporated into electrocompetent TOP10F′ E. Coli (Invitrogen) usingstandard techniques (see Current Protocols in Molecular Biology, section1.8.4). A test transformation yielded 5×10⁹ transformants per pg of pUCDNA. After transformation, the transformation efficiency was determinedby plating dilutions onto LB-amp plates (100 μg/ml ampicillin) andcounting surviving colonies. For the library insert generated from OL-2,a 4:1 insert:vector molar ration in the ligation gave a transformationefficiency of 3.98×10⁷ transformants per pg vector DNA used in theligation, with a large scale transformation efficiency of 4.8×10⁷transformants per ug vector. The vector alone ligation generated 40 foldfewer transformants. 10 colonies from the transformation with the OL-1insert ligation were picked, cultured and the DNA prepared and sequencedto identify the correct clone.

[0208] The remainder of the OL-2 library SOC/transformation mixture wasinoculated into 500 μl LB-amp (100 μg/ml ampicillin) and incubated at37° C. with shaking (300 rpm). The Abs₆₀₀ of the library culture wasmonitored. When the culture reached an Abs₆₀₀ of 0.8 (approximately fivehours), 100 μl were removed, pelleted, resuspended in 10 ml LB/15%glycerol and stored in 1 ml aliquots at −80° C. (An Abs₆₀₀ of 0.8 equalsa cell concentration of approximately 10⁹ cells per ml. Therefore, for alibrary of 4.8×10⁷, each frozen aliquot will contain 200 libraryequivalents).

[0209] Analysis of Library Diversity

[0210] Surviving colonies plated above were screened by PCR with primersflanking the degenerate region to determine the fraction of clones whichcontained insert (>90%). 8 insert-containing clones were picked and thenucleotide sequences of the degenerate and flanking non-degenerateregions determined. Each nucleotide was represented in the N positionswith approximately 25% frequency, while G or T (but not A or C) wasrepresented in the K positions with approximately 50% frequency. Thefrequency of stop codons generated in the degenerate region can bedetermined by this method as well.

[0211] Generation of Library Retrovirus and Infection of Jurkat Cells

[0212] DAY 0: Preparation of Phoenix Retrovirus Producer cells forTransfection: 18-24 hours prior to transfection, Phoenix cells wereevenly plated at 1.5-2 million cells per 60 mm plate in Producer cellgrowth media (DMEM: 10% FCS, 1% Penicillin-Streptomycin, 1% Glutamine).Cells were allowed to attach for 20 hours on the plates.

[0213] DAY 1: Transient transfection: The highest transfectionfrequencies are obtained with Phoenix cells that are 70-80% confluent atthe time of transfection. The DNA in HBS (2×HBS=8.0 g NaCl, 6.5 g HEPES,10 ml Na₂HPO₄ stock (5.25 g dibasic in 500 ml water), adjusted to pH 7,to a final volume of 500 mls, with a final pH adjustment to 7) wasprepared for application to the Phoenix cells. About 5 minutes prior totransfection, chloroquine (Sigma) was added to each plate to 25 uM(chloroquine stock is 50 mM in ddH2O; for 3 mL media+1 ml DNA, add 2μl). To a 15 ml conical tube, the following were added (per 6 cm plate,5 plates total, with all reagents at room temperature):

[0214] 5 ug library DNA (DNA was added in a drop to side of tube)

[0215] 1 ug pMSCVpc lacZ virus vector

[0216] 438ul dd H2O (the DNA was washed to the bottom of tube withwater).

[0217] 61 ul 2M CaCl₂ (Mallinkrodt, catalog #4160; make up in water,sterile filter and store tightly capped at 4° C.

[0218] 500 ul Total volume.

[0219] Samples were mixed thoroughly with finger tapping. Transfectionswith 5 ug pMSCVpc lac Z and with the OL-1 vector DNA were carried outfor use as negative and positive controls, respectively. 0.5 mL 2×HBSwas added to each tube quickly; the solution was bubbled vigorously withthe automatic pipettor by keeping the eject button depressed) for 10 sec(the actual length of bubbling time depends on each batch of 2×HBS). TheHBS/DNA solution was dispersed dropwise and evenly onto the media ineach Phoenix cell plate dropwise (gently and quickly). The plates wereobserved under a microscope; evenly distributed very small blackparticles of precipitated DNA (like pepper) were visible. The plateswere placed in a 37° C. incubator and rocked forward and backward a fewtimes to evenly distribute the DNA/CaPO4 particles. 6-8 hourspost-transfection, the media was changed to 3 ml fresh DMEM, 10% FCS.Prior to the media change, the DNA precipitate was larger and moreclearly visible under the microscope.

[0220] DAY 2: Second media change.

[0221] 24 hours post-transfection, the media was changed again to 3 mlfresh DMEM, 10% FCS. The cells were placed at 32° C. (the virus is morestable if incubation is carried out at 32° C., although 37° C. is fine).

[0222] DAY 3: Transduction of Jurkat EcoR cells.

[0223] A sterile Acrodisc 0.45 micron syringe filter (Gelman Sciences)was attached to the end of a 10 ml sterile syringe and the injectionstopper sterilely removed from the syringe barrel. At 48 hourspost-transfection, the virus supernatant was removed from the Phoenixcells and added to the syringe barrel. The stopper was replaced and thevirus supernatant was ejected dropwise into a clean, sterile conicaltube. The Phoenix cell plates were set aside for X-Gal staining (seebelow). Polybrene was added to each viral supernatant (Sigma; 2.5 mg/mlin ddH20=500×; store at −20° C.) to a final concentration of 5 mg/ml.4.5×10⁶ Jurkat EcoR cells (Jurkat cells stably expressing the ecotropicretrovirus receptor) were pelleted for 1400 rpm for five min andresuspended in 9 mls of the OL-2 library virus supernatant. The cellswere distributed in aliquots of 1 ml, or 5×10⁵ cells, into the wells ofa 24 well plate. 1.5×10⁶ Jurkat EcoR cells were similarly treated with 3mls each of the lacZ viral supernatant and the OL-1 viral supernatant.Each cell plate was wrapped in parafilm, placed in a microplate carrier(DuPont) and centrifuged at 2500 rpm for 90 min at 32° C. in aDuPont/Sorvall RT 6000B table top centrifuge. After centrifugation, thecells were observed under a microscope. The presence of largeirregularly-shaped bodies representing fused Jurkats (each as large as5-10 unfused cells) suggested successful infection. The parafilm wasremoved from the plates, which were placed at 32° C. After an additional16 hours at 32° C., the cells were loosened from the bottom of each wellwith gentle trituration and added to a 15 ml conical tube. The tubeswere centrifuged at 1400 rpm for five min to the pellet the cells. Thecells were resuspended in 5 mls fresh RPMI,10% FCS for every three wellsof cells and added to a 60 mm plate (3 wells of cells per plate). 1 mlfresh RPMI, 10% FCS was added to each well of cells remaining in the 24well plates. Plates were kept at 37° C. for 72 hours, at which time thecells transduced with each virus were combined and an aliquot Jurkatcells stained with X-Gal. Unused viral supernatant was stored at −80° C.for future transduction, although the titer drops by one-half for eachfreeze-thaw cycle.

[0224] Determination of Transfection Efficiency

[0225] Both tranfected Phoenix cells and transduced Jurkat cells werestained with X-Gal to gauge the transfection and transductionefficiencies. The purpose of co-transfecting the pMSCVpc lacZ virusvector with the library virus vector, as described above, was to permitan indirect assessment of the efficiencies of transfection andtransduction. Preparation of solutions: fixative: PBS/0.10%Glutaraldehyde. Glutaraldehyde stock (Sigma cat # G5882) is a 25%solution, or 250×; stock staining solutions: i) 300 mM/25×ferrocyanatesolution: 25.3 g K4Fe(CN)6.3H2O (Mallinckrodt)+2.48 g MgCl2 (Sigma) in200 ml H2O; store at 4° C.; ii. 300 mM/25×ferricyanate solution: 19.75 gK3Fe(CN)6 (Sigma)+2.48 g MgCl2 in 200 ml H2O; store at 4°; iii. XGal(Molecular Probes) is made up as a 40 mg/ml solution in DMF; store at−20° C. in the dark; iv. 1×ferro/ferricyanate solution: add 4 ml 300mM/25×ferrocyanate solution and 4 ml 300 mM/25×ferricyanate solution to196 ml PBS; store at 4° C. for up to one month; v. active stainingsolution: each time cells are to be stained, 100 μl 40 mg/ml X-Gal isadded to each 3 ml 1×ferro/ferricyanate solution; washing solution: PBSfor Phoenix and other adherent cells; 1% FCS in PBS for Jurkat and othernonadherent cells.

[0226] The media was removed from the 60 mm plates of Phoenix cells or5×10⁵ Jurkat cells were pelleted in a 15 ml conical tube at 1400 rpm forfive min. 2 ml of fixative were added to each 60 mm plate of Phoenixcells or Jurkat cells were resuspended in 1 ml fixative. Cells were leftin fixative for 2 min. For Phoenix cells, fixative was poured off andthe cells were washed three times with PBS (first two washes were quick;for third wash, the PBS was left on the cells for 3 min). For Jurkatcells, the fixative was quenched by adding 5-10 ml PBS/1% FCS to eachconical tube, inverting each tube five times and pelleting as before. 3ml of active staining solution were layered onto each 60 mm plate ofPhoenix cells or each cell pellet of 5×10⁵ Jurkat cells was resuspendedin 1 ml of active staining solution and placed in a well of a 24 wellplate. All cells were incubated at 37° C. The cells were observed undera microscope 24 hours later. The efficiency of transfection of thePhoenix cells was estimated as the percentage of blue cells in a field.The efficiency of transduction of the Jurkat cells was estimated bycounting blue cells in a hemocytometer. Transfection with 5 μg lacZvector produced 50% blue Phoenix cells. Transduction of Jurkats with theresulting virus produced 30% blue Jurkat cells. Co-transfection of 1 μglacZ virus vector with 5 μg library virus vector produced 5-10% bluePhoenix cells. Transduction of Jurkats with the resulting virus resultedin 3-10% blue Jurkat cells.

[0227] Selection of Jurkat Cells with IgM Anti-Fas

[0228] Titer IgM anti-Fas: A fresh batch of CH-11 IgM antibody to humanFas (Kamiya Biomedical Company; cat # MC-060)was tested to determine theeffectiveness of induction of apoptosis. 5×10⁵ Jurkat EcoR cells werepelleted at 1500 rpm for five min and resuspended in 1 ml RPMI/2.5% FCSplus serial dilutions of CH-11 antibody, 50 ng/ml, 10 ng/ml, 2.0 ng/mland 0.5 ng/ml final concentration. Cells in each dilution of antibodywere placed in a well of a 24 well plate at 37° C. for 48 hours, atwhich time 4 ml acridine orange/ethidium bromide (Sigma; 100 μg/ml eachin PBS; store in the dark at 4° C.) was added to 100 ml cells on ice.Cells were examined in a hemocytometer under a 20×objective with afilter combination suitable for reading fluorescein.

[0229] 2. 100 cells from each sample were counted and the number ofcells in the following groups was recorded:

[0230] 1. live cells with normal nuclei (bright green chromatin withorganized structure).

[0231] 2. early apoptotic (EA; bright green chromatin that is highlycondensed or fragmented).

[0232] 3. late apoptotic (LA; bright orange chromatin that is highlycondensed or fragmented).

[0233] 4. necrotic cells (N; bright orange chromatin with organizedstructure). % apoptotic cells was calculated as EA+LA/total number ofcells counted×100 Using 10 ng/ml of the CH-11 antibody, >95% apoptosisof Jurkat EcoR cells was demonstrated.

[0234] IgM Anti-Fas Selection of Library-expressing Jurkats

[0235] 9.6×10⁶ OL-2 library-transduced Jurkat cells were pelleted andresuspended in 96 ml RPMI/2.5% FCS+10 ng/ml CH-11 antibody. Cells weredistributed in 1 ml aliquots of 1×10⁵ cells into each well of four 24well plates. 4.8×10⁶ lacZ-transduced Jurkats and OL-1 -transducedJurkats were similarly treated and each distributed into the wells oftwo 24 well plates. Plates were placed at 37° C. for five days. Theplates were checked daily for bacterial or yeast contamination. Cellswere removed from any contaminated wells and 2 ml 10 N NaOH was added tothe empty wells to reduce the risk of spread of contamination to otherwells. Little to no live cells were observed under the microscope after2-3 days, confirmed by the red color of the media which had not beendepleted of any nutrients. Five days after initial IgM anti-Fastreatment, 1 ml RPMI/20% FCS was added to each well. The cells were leftat 37° C. for an additional 10-14 days. The plates were checkedfrequently for contamination and treated as above. 10 days afteraddition of the RPMI/20% FCS, nearly every well of the OL-1-transducedcells contained live colonies of cells, confirmed by the orange color ofthe nutrient-depleted media. The media in all wells of lacZ-transducedcells remained red, and little cell growth was observed in any of thewells. Selected wells of the OL-2 library-transduced cells containedlive cells and nutrient-depleted media. During the next two weeks, cellswere removed from all wells in which significant cell growth wasoccurring, as gaged by observing the cells directly under the microscopeand monitoring the increasing nutrient depletion of the cell media.Cells from each well were resuspended in 5 ml fresh RPMI/10% FCS andplaced in a 60 mm dish at 37° C. for 2-3 days.

[0236] RNA Isolation

[0237] RNA was isolated from the each surviving well population of OL-2library-transduced Jurkat cells (17 wells), as well from five survivingwell populations of OL-1-transduced cells, using the mRNA Capture Kitaccording to the manufacturer's protocol (Boehringer-Mannheim cat #1 787896). Briefly: 5×10⁵ cells from each dish were pelleted at 1400 rpm forfive min in an Eppendorf tube, washed twice with PBS and resuspended in200 ml lysis buffer and sheared by passing six times through a 21 guageneedle attached to a 1 ml syringe. 4 ml 1:20 dilution of biotinylatedoligo(dT)20 was added to each sample and incubated for 3 min at 37° C.The mix was removed from each tube. Each tube was washed three timeswith 200 ml of washing buffer. Cells were also stored in 90%FCS/10%DMSOin 1 ml aliquots of 1×106 cells each in liquid nitrogen.

[0238] RT PCR Rescue of Peptide-encoding Inserts from Selected Cells

[0239] PCR was carried out using the TitanTM RT-PCR System (BoehringerMannheim cat #1 855 476), using two primers: 5′pBL primer has thesequence: 5′-GAT CCT CCC TTT ATC CAG-3′ (SEQ ID NO:64) and iscomplementary to nucleotides 1364-1381 of all pMSCVpc-based vectors andretrovirus mRNA, just upstream of the cloned insert. 3A primer has thesequence 5′-CTA CAG GTG GGG TCT TTC-3′ (SEQ ID NO:65) and iscomplementary to a sequence in all pMSCVpc-based vectors and retrovirusmRNA, just downstream of the cloned insert.

[0240] Re-cloning Rescued Peptide-encoding Inserts

[0241] Each PCR-rescued sample was extracted with phenol chloroform,ethanol precipitated and resupsended in 25 ml 10 mM Tris pH 8.5. 3 mlnondenaturing DNA gel loading dye was added to 10 ml of each sample andrun on a 10% acrylamide minigel with oligonucleotide quantitationstandards and a 10 base pair ladder, as described above. Each lanecontained one prominent band with the expected molecular weight of 216base pairs and minor background bands. The molarity of each sample wasquantitated using NIH Image as before. Each sample was BstXI restrictiondigested, phenol extracted, ethanol precipitated and resuspended in 25ml 10 mM tris pH 8.5. The purified samples were loaded onto 10%acrylamide gels and quantitated as before. All samples contained aprominent band of 55 base pairs, the expected molecular weight for therestriction digested insert, as well as bands of 100 base pairs and 51base pairs corresponding to each of the ends of the rescued DNA insertremoved by the restriction enzyme. Each restriction digested,PCR-rescued insert was ligated at a 4:1 insert:vector molar ratio with100 ng pMSCVpc vector DNA, precipitated and electrotransformed asbefore. Surviving colonies for each transformation were PCR screenedusing the 5′pBL and 3A primers. 8 to 10 insert-containing colonies foreach transformation were cultured overnight, the cultures were pooledand a single mini-DNA preparation carried out for each pool.

[0242] Fas-Selected Peptide clones: All peptides have the sequence: METGLY LYS ARG LYS GLY XXX XXX XXX D/E XXX VAL GLY PRO PRO (SEQ ID NO:62).Only the xxx xxx xxx D/E xxx (SEQ ID NO:100) amino acids are writtenabove each DNA sequence below.

[0243] From first library selection well: L1B3 INDIVIDUAL CLONES,FAS-SELECTED.                                THR ALA SER ASP ALA (SEQ IDNO:67) L1B3E1 ATG GGC AAG AGA AAG GGC ACG GCG TCT GAT GCT (SEQ ID NO:66)GTG GGG CCC CCT TAA                                TYR PRO SER ASP VAL(SEQ ID NO:69) L1B3E2 ATG GGC AAG AGA AAG GGC TAT CCT TCT GAT GTG (SEQID NO:68) GTG GGG CCC CCT TAA                                THR PRO SERASP MET (SEQ ID NO:71) L1B3E3 ATG GGC AAG AGA AAG GGC ACG CCT TCG GATATG (SEQ ID NO:70) GTG GGG CCC CCT TAA                               THR ALA SER ASP LEU (SEQ ID NO:73) L1B3E6ATG GGC AAG AGA AAG GGC ACG GCT TCT GAT CTT (SEQ ID NO:72) GTG GGG CCCCCT TAA                                SER ASP ARG ASP ILE (SEQ IDNO:75) L1B3E7 ATG GGC AAG AGA AAG GGC TCT GAT AGG GAT ATT (SEQ ID NO:74)GTG GGG CCC CCT TAA From second library selection well: L2A5 INDIVIDUALCLONES, FAS SELECTED.                                TRP LEU LEU GLU PHE(SEQ ID NO:77) L2A5A2 ATG GGC AAG AGA AAG GGC TGG TTG CTA GAG TTT (SEQID NO:76) GTG GGG CCC CCT TAA                                TRP LEU ILEGLU PHE (SEQ ID NO:79) L2A5A3 ATG GGC AAG AGA AAG GGC TGG TTG ATA GAGTTT (SEQ ID NO:78) GTG GGG CCC CCT TAA                               TRP LEU LEU GLU PHE (SEQ ID NO:77) L2A5A6ATG GGC AAG AGA AAG GGC TGG TTG CTA GAG TTT (SEQ ID NO:76) GTG GGG CCCCCT TAA                                TRP LEU LEU GLU PHE (SEQ IDNO:77) L2A5A8 ATG GGC AAG AGA AAG GGC TGG TTG CTA GAG TTT (SEQ ID NO:76)GTG GGG CCC CCT TAA                                SER TYR GLN ASP LEU(SEQ ID NO:81) L2A5A9 ATG GGC AAG AGA AGA GGC TCT TAC CAA GAT CTG (SEQID NO:101) GTG GGG CCC CCT TAA

Example 3 Staurosporine Selection of NIH 3T3 Cells Transduced with pBabePuro Peptide Library

[0244] A. Library construction. Construction of the pBabe puro randompeptide library was described earlier in the patent. The randomizedpeptide has the sequence: MGXXXXXXXXXXGGPP (SEQ ID NO:82). The diversityof the library is 2×10⁸ at the DNA insert level.

[0245] B. Library transfection. Transfections were carried out asdescribed for Fas-selection, but in 15 cm plates of 10⁷ Phoenix cells.The DNA solution added to each plate consisted of: 50 ug library DNA, 5ug lacZ vector, 4340 ul ddH₂O, 610 ul 2M CaCl₂ and 5000 ul 2×HBS.

[0246] C. Library transduction. 24 hours prior to transduction, 2×10⁷NIH 3T3 cells were plated in each of ten 15 cm plates in 25 ml DMEM, 10%Bovine Calf Serum. 5 ml library virus supernatant was added to eachplate (plus polybrene as before). 24 hour after transduction, media waschanged to 25 ml fresh DMEM, 10% BCS. Cells were stained with X-gal at48 hours post-transduction. The transduction efficiency was estimated as40-50%.

[0247] D. Staurosporine selection. Staurosporine, an alkaloid fromStreptomyces sp., is a potent, broad spectrum inhibitor of proteinkinases which binds the ATP site. Addition of 1 uM staurosporine inserum-free media to NIH 3T3 cells induced >99% apoptosis within 24hours, as determined by ethidium bromide/acridine orange double stainingas described for the Fas selection.

[0248] 2×10⁶ library-transduced NIH 3T3 cells were plated in each of 1015 cm plates. Cells were allowed to attach for 24 hours, at which timestaurosporine was added to 1 uM in serum free DMEM. LacZ-transduced NIH3T3 cells and BCL-2-transduced NIH 3T3 cells were used as negative andpositive controls, respectively. 24 hours after staurosporine treatment,the media was changed to 25 ml fresh DMEM, 10% BCS. The media waschanged every two days for one week, until the surviving cells lookedhealthy (typical 3T3 morphology), at which time 1 uM staurosporine inserum-free media was added again. The media wash changed to DMEM, 10%BCS as before. Stp treatment was carried out again for a total of threetreatments, at which time the number of library-transduced cellssurviving appeared greater than the number of lacZ-transduced cells (butless than the BCL-2-transduced cells.

[0249] E. Moloney transfer. After the second staurosporine treatment,aliquots of surviving cells from each plate were infected with wild typeMoloney murine leukemia virus supernatant. (generated by transfectingPhoenix cells with the retroviral vector pZap). The virus was allowed tospread through the culture for one week (with re-plating of the cellsevery 2-3 days). Cells were plated as before and treated withStaurosporine before proceeding to RNA isolation and PCR rescue.

[0250] F. RNA isolation. Aliquots of cells surviving in each plate wereresuspended in 90% FCS, 10% DMSO and stored in liquid nitrogen. RNA wasprepared with Trizol reagent (Gibco BRL, cat# 15596-026). Briefly, 1 mlTRIzol reagent was added were 10 cm² monolayer of cells and incubatedfor 5 min at room temperature. Cell lysates were transferred to 15 mlconical tubes (Note: at this point, DEPC-treated solutions and glasswarewere used exclusively). 0.2 ml chloroform was added per 1 ml TRIzolreagent used. Tubes were shaken for 15 sec, incubated for 3 min at roomtemperature and centrifuged at 12000×g for 15 min at 4° C. TheRNA-containing upper aqueous phase was removed and 0.5 ml isopropanoladded per 1 ml TRIzol used for the initial homogenization. Samples weremixed and incubated at room temperature for 10 min followed bycentrifugation as before. the supernatant was removed and the RNA pelletwashed with 75% ethanol (1 ml per 1 ml TRIzol). The sample was vortexedand centrifuged at 7500×g for 5 min at 4° C. The RNA pellet wasair-dried for 10 min and resuspended in RNase-free water with 10 minincubation at 60° C. to dissolve the pellet. RNA concentration wasdetermined by measuring the absorbance at 260 nm.

[0251] G. PCR rescue. PCR rescue was carried out as for Fas selection,using the primers 5′pBL and SV 40 down. The second primer has thesequence: 5′ CTG ACA CAC ATT CCA CAG 3′ (SEQ ID NO:83) and iscomplimentary to positions 1424-1441 of the pBabe Puro retroviralvector. PCR reactions were extracted with phenol-chloroform,precipitated with ethanol and digested with Bam HI and Sal I beforeligation with the retroviral vector pWZL neo. The figure shows a 10%acrylamide gel of representative PCR-generated inserts:

[0252] Lane 1: 10 base pair ladder

[0253] Lane 2: undigested PCR insert from Stuarosporine-selected cellpopulation

[0254] Lane 3: undigested PCR insert from same cell population, afterMoloney rescue and Staurosporine selection.

[0255] Lanes 4 and 5: same as lanes 2 and 3, after restrictiondigestion.

[0256] H. Secondary screen. pWZI neo vectors containing rescued insertswere transfected into Phoenix cells, and the resulting virus used totransduce NIH 3T3 cells. Staurosporine selection was repeated threetimes as before, before RNA preparation and PCR rescue.

[0257] I. Sequences of the first 9 positives:

[0258] The sequences of the first nine positives are as follows:SEQUENCE OF 2 P 1 GGATCCAGTGTGGTGGTACGTAGGAATACC- (SEQ ID NO:84) ATG GGATGT CCG TCT GTT GCT AGG CCG CGG GGT GGT GGG GGC CCC CCC (SEQ ID NO:85)Met Gly Cys Pro Ser Val Ala Arg Pro Arg Gly Gly Gly Gly Pro ProTAGCTAACTAAAGATCCCAGTGTGGTGGTACGTAGGAATTCGCC 2P1 Stp Stp Stp Bam /BgSEQUENCE OF 4 P 1 GGATCCCAGTGTGGTGGTACGTAGAATACC- (SEQ ID NO:8~) ATG GGATTG TCT TTT GTT ATT (C/TGT CTG CAG CAT CGT GGG GGC CCC (SEQ ID NO:87)Met Gly Leu Ser Phe Val Ile Arg   Leu Gln His Arg Gly Gly Pro CCC TAGCTAACTAAAGATCCCAGTGTGGTGGTACGT 4P1 Pro Stp  Stp StpBam /Bg      CysSEQUENCE OF 5 P 1 GGATCCCAGTGTGGTGGTACGTAGGAGTACC- (SEQ ID NO:58) ATGGGA CCT CCG ATT TGG TAT ACT CAT TGG AGT CAT GGG GGC CCC CCC (SEQ IDNO:89) Met Gly Pro Pro Ile Trp Tyr Thr His Trp Ser His Gly Gly Pro ProTAG CTAACTAAAGAT CC 5P1 Stp Stp StpBam /Bg SEQUENCE OF 6 P 1GGATCCCAGTGTGGTGGTACGTAGGAGTACC- (SEQ ID NO:91) ATG GAA GTC AGG CGT TTGTGA ATA CTC GGC ATA AG GGG GGC CCC CCC (SEQ ID NO:3) Met Glu Val Arg ArgLeu Stp                    Gly Gly Pro Pro (SEQ ID NO:90)TAGCTAACTAAAGAT CC 6P1 Stp Stp StpBam /Bg SEQUENCE OF 7 P 1CCGGCCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGAT (SEQ IDNO:92) CTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAACGTCTAGGCCCCC7P1 SEQUENCE OF 8 P 1 GGATCCCAGTGTGGTGGTACGTAGGAATACC (SEQ ID NO:93) ATGGGA CTT TAG CCG GGC CCC CCCTAGCTAACTAAAGATCCCAGTGTGGTGGT Met Gly LeuStp         Pro Pro Stp Stp Stp Bam /Bg ACGTAGGAATTCGCCAGCACAG T 8P1SEQUENCE OF 9 P 1 GGATCCCAGTGTGGTGGTACGTAGGAATAC (SEQ ID NO:95) ATG GGAACT GTT ATG GCG ATG TCG GAT TAG GTC GAG GGG GGC CCC CCC (SEQ ID NO:3)Met Gly Thr Val Met Ala Met Ser Asp Stp         Gly Gly Pro Pro (SEQ IDNO:94) TAGCTAACTAAAGATCC 9P1 Stp Stp Stp Bam /Bg SEQUENCE OF 10 P 1GGATCCAGTGTGGTGGTACGTAGGAATACC (SEQ ID NO:96) ATG GGA TGT CCG TCT GTTGCT AGG CCG CGG GGT GGT GGG GGC CCC CCC (SEQ ID NO:97) Met Gly Cys ProSer Val Ala Arg Pro Arg Gly Gly Gly Gly Pro Pro TAGCTAACTAAAGATCC 10P1Stp Stp Stp Bam /Bg

Example 4 Use of NF-κB and NFAT in Signalling

[0259] The NFκB/IkB complex is the classic pro-inflammatory secondmessenger system, known to be involved as a positive regulator of anumber of pro-inflammatory processes and cytokines. These include, butare not limited to, IL-1, IL-6, IL-8, and TNF-α. As well,anti-inflammatory interleukins, such as IL-4, can lead to directdown-modulation of NF-kB in synovial fibroblasts and concomitantdownregulation of IL-6 production. The NF-kB/IkB complex is awidespread, acute-phase, rapid-response transcriptional activationsystem. It operates in most cell types tested, but leads to differentoutcomes dependent upon the cell type and the nature of the initiatingstimulus. Activators of NF-kB include LPS, TNF-α, IL-1, inducers of Tcell activation, protein synthesis inhibitors, phorbol esters, anda-IgM. Other inducers include the viruses Adenovirus, HTLV I,cytomegalovirus, Sendai, and Herpes simplex I, agents that causecellular damage such as ultraviolet light and peroxides, and phosphataseinhibitors such as okadaic acid. These inducers act through PKA andPKC-dependent pathways, double-strand RNA-dependent kinase, and otherpathways. Pharmacologic regulators of NF-kB, such as salicylate andglucocorticoids, act by either preventing IkB-α degradation or lead toupregulation of IkB-a transcription and steady-state levels, therebyacting to prevent the activation of this critical factor.

[0260] NF-kB (Nuclear Factor that binds to the k locus B site) ispresent in the cytoplasm of most cells in an inactive form complexed toIkB (Inhibitor of NF-kB). Certain stimuli received by cells areprocessed by cellular signaling mechanisms and integrated in thespecific phosphorylation of IkB and its degradation. The regulation ofIkB-a function is through a Signal Response Element (SRE) in the aminoterminus of the molecule. Phosphorylation of serine residues 32 and 36leads to recognition of the IkB-a molecule by the ubiquitinationmachinery, release of NF-kB to the nucleus, and degradation of IkB.Therefore, dependent upon the phosphorylation/degradative state of IkB,NF-kB is either maintained in the cytoplasm or released to the nucleus.In the nucleus NF-kB binds to a consensus DNA motif found near theregulatory regions of many characterized genes and therein acts as atranscriptional regulator. Importantly, from the point of view ofinfectious disease, NF-kB is a primary activator of the HumanImmunodeficiency Virus (HIV). Suitable induced genes include TNF-α andIL-6.

[0261] Biochemically, NF-kB is defined as a heterodimer of twopolypeptides, p50 and p65, of corresponding molecular mass 50 and 65 kD,respectively. p50 is processed from a 105 kD precursor protein by an asyet uncharacterized mechanism. p65 is the receptor for IkB and is themolecule through which IkB exerts its inhibitory/regulatory effects onNF-kB. These are the prototypic transcription factors that define alarge family of classical Rel/NF-kB factors.

[0262] Cloning of the p50 and p65 components of NF-kB led to thediscovery of a family of related factors, termed Rel. Both p50 and p65have a 300 amino motif (Rel) at their amino termini that was originallydescribed in the proto-oncogene c-rel and the Drosophilaaxis-determining gene, Dorsal. The family of polypeptides revealed byp50 and p65 have overlapping DNA-binding specificities, differentialtissue distribution, and complex regulatory phenomena. p105(p50) isrepresentative of the ankyrin-motif-containing Rel proteins that areprocessed in the cytoplasm to smaller proteins lacking the carboxylterminus. The carboxyl terminus of p105 shows structural and functionalhomologies to IkB (which also has ankyrin motifs) and functions with anIkB-like activity both in cis and in trans. p65 is representative of asecond group of Rel proteins that have divergent carboxyl termini—theseregions have been suggested to encode transcriptional activationdomains. The 300 amino acids of Rel domains manifest four importantfunctions: 1) DNA-binding in the roughly amino-terminal ⅓ of the domain,2) dimerization in the carboxyl portion of the domain, 3) interactionwith ankyrin-containing IkB-like proteins, and 4) nuclear-localizingsignal at the carboxyl terminus of the Rel domain. In p50 the Rel domainalso includes a transcriptional activation domain.

[0263] NFAT, the Nuclear Factor of Activated T Cells (NFAT), is theimmediate early acute phase response factor for T cell activation.Inhibition of NFAT by cyclosporin A (CsA) leads to blockade of IL-2production and loss of T cell commitment to activation. NFAT, a criticalcomponent of pro-inflammatory events carried out by T cells, is also thefactor blocked by CsA in transplantation. Upon cloning NFAT it was clearcontains a region of the molecule implicated in DNA binding that hassignificant homology to the Rel family of proteins. Based on structuralconsiderations, homology comparisons, and similar modes of action, aswell as genomic structures of the molecules indicate similar intron/exonboundaries in NF-kB and NFAT families, thus indicating that NFATactually belongs to the Rel family of factors by lineal descent and thatits interaction with pro-inflammatory transcriptional regulators of thebZIP family would follow a general set of rules common to the NF-kB/bZIPinteractions.

[0264] We have shown that NFAT is involved in pro-inflammatory responseto mitogens in activation of HIV-1 (S. Kinoshita and G.P.N, submitted)and that the binding of NFAT to sites overlapping the NF-kB sites ofHIV-1 is responsible for this process. This work follows on work byothers showing that NFAT can regulate TNF-a activation in interactionwith ATF-2/Jun and GM-CSF. Interestingly, NFAT also appears to beinvolved in regulation of mast cell release of IL-4, an importantregulator of pro-inflammatory cytokines, such as IL-1β, TNF-α and IL-6.The activity of NFAT in these systems has all shown to bepharmacologically modulated by CsA. Thus, although NFAT was originallydiscovered as a T cell specific factor, it was later found to beresponsible for a host of immediate early, acute phase responseactivities, as well as direct regulation of IL-4.

[0265] Therefore, the extended Rel families of NF-kB and NFAT makeattractive targets for inhibition and modulation of pro-inflammatoryaction. Their involvement in numerous regulatory pathways and theirdecisive roles in such processes, including the specific interactionsthey elaborate with bZIP proteins, make them attractive specific targetsfor inhibition.

[0266] Reporter Genes for Detection of TNF-α and IL-1 Promoter Activity

[0267] We designed a retrovirus-based luciferase reporter-gene systemdriven by a minimal promoter and two lgk NF-kB sites. In the constructspresented here, the deletions I introduced were more extensive thanthose previously published, since preliminary experiments showed thatresidual enhancer activity resided in commonly available deletionconstructs (Nolan, Saksela and Baltimore, unpublished). The vectorsdesigned were pSinII-luc (containing a luciferase gene in the retroviralsense orientation to test for residual promoter activity in theconstruct backbone), pSinII-fosluc (identical to pSinII luc exceptcontains a minimal fos promoter element to test for residual enhanceractivity in construct backbone), and pSinII-2kBfosluc (derived frompSinII-fosluc with 2 Igk kB sites cloned 5′ proximal to the fos minimalpromoter as a reporter for NF-kB activity). These three vectors used toinfect 1×10⁶ 70Z/3 cells. 70Z/3 is a murine pre B cell line originallyused in the initial characterization of NF-kB. After 48 hours, theinfected cells were split into two fractions (stimulated with LPS andunstimulated). Six hours later, cell extracts were prepared and assayedfor luciferase activity (extract representing ˜10⁴ cells was used foreach point). The results showed that SinII-luc showed no indiction,SinII-fosluc showed roughly a one-fold increase, and SinII-2kBfoslucshowed a four fold induction in lucerifase activity. Accordingly,retrovirally based reporter constructs can be used to sensitively reportNF-kB activity in native chromatin. It now becomes possible to combinereporter gene technology with retroviral delivery of effector peptides.Unstimulated cells and stimulated controls (uninfected and SinII-luc)showed little or no activity. Importantly, then, retroviral delivery didnot result in significant background induction of NF-kB activity, aproblem with other transfection procedures. The SinII-luc andpSinII-fosluc controls shows no significant residual promoter orenhancer activity in the construct. No significant readthrough fromendogenous genomic loci or endogenous enhancer activity that mightobscure readings was detected. These latter results are consistent withprevious work using gene search retroviruses employing lacZ and flowcytometry. In these studies less than 0.1% of random integration eventsshowed endogenous cis-regulation of the integrated constructs.

[0268] These construct designs will be used as the basis for rapidcreation and testing of TNF-α and IL-1 promoter studies in T cells,macrophages, and synovial cells. We will incorporate in the place ofluciferase either the lacZ or GFP cDNAs for FACS-based assay. We willplace up to three to four kilobases of TNF-α or IL-1 promoter region inplace of the minimal promoter employed here. These constructs will beused as a proxy measure of endogenous TNF-α and IL-1 promoter activityand will serve to allow for searches for peptides from our librariesthat act upon NF-kB or NFAT as well as unknown signaling pathways thatare independent of NF-kB or NFAT critical to TNF-α and IL-1 signaling.

[0269] The B cell lines to be used are 70Z/3. T cells to be used arehuman Jurkat. Macrophage lines to be used are Raw 309 and the P388D1line which is highly responsive to PMA induction of secreted IL-1.Synovial cells to be used are HIG-82 and can be activated with IL-1 toinduce metalloproteases and with TNF-α to induce NF-kB. IL-1 inductionof metalloproteases acts through NF-kB on collagenase and othermetalloproteases of this group. Thus, we have shown that β-gal fused toIkB-α and delivered via a retrovirus to cells responds to stimuli thatdegrade IkB-α as follows: a) 70Z/3 pre-B cells were infected with aretrovirus expressing a fusion of β-gal to either wild-type IkB-α or aninactive, dominant negative IkB-α; infection efficiency wasapproximately 30%. Cells were stimulated with LPS for varying times andthen loaded with FDG for measure of b-gal expression by FACS. b) Cellsfrom (a) were induced for maximal LPS induction of IkB-α degradation andtreated with either salicylate or control. Salicylate blockeddegradation of the β-gal-IkB fusion to the same extent as the dominantnegative IkB-α.

[0270] Direct Detection in Living Cells of Steady State Levels of IkB-α

[0271] At the first approach, NF-kB activation will be measured usingour newly developed IkB-α mobile reporter system described above. Inthis approach, the N-terminus of IkB-α has been translationally fused tothe lacZ gene. In mammalian cells, β-galactosidase expression can bemeasured using the Fluorescence activated Cell Sorter (FACS) on a cellby cell basis. By coupling β-gal to IkB-α, the stability of β-gal isfunctionally dependent upon IkB-α. Since signals in cells that activateNF-kB lead to the degradation of IkB, β-gal was similarly degraded; asabove, cells were infected with a retrovirus containing a β-gal-IkB-αfusion and induced them with stimuli that lead to activation of NF-kB.We can use the cell sorter to distinguish cells that have degraded IkB-αon a REAL-TIME basis, and not through activation of proxy reportergenes. These lines were shown to respond accordingly after treatmentwith the anti-inflammatory agent salicylate (aspirin) which has beenshown to be a direct inhibitor of NF-kB activation. We have used thisand related protocols in B cells to select for novel mutants of IkB-αand have thereby defined new regions of the IkB-α molecule that respondto differential signaling (J. Caldwell and G. Nolan, unpublished).

[0272] 1×10⁷ cells carrying the reporter will be infected at highefficiency with the molecular libraries described herein. Cells will bestimulated with LPS, TNF-α, IL-1 or PMA, and then used to select by FACSfor those cells that DO NOT degrade β-gal. After growing out of thecells, the population will be restimulated as before and sorted again.Cells will be sorted until the population is 100% heritable for the lackof degradation phenotype. Inserts will be rescued, recloned into aretrovirus construct, and then screened again until a trans-phenotypecan be confirmed. Peptides will be sequenced as noted.

[0273] Selection NFAT-deficiency Using Cell-death Induction by NFATDependent Pathways

[0274] We have devised a system for selecting for blockade of NFATsignaling in cells that can be employed with our retroviral libraries.The system is based upon findings by Serafini and colleagues in whichthey were able to create a cell line whose death was dependent uponactivation of NFAT. Cells stimulated by activators of T cells or NFATlead to activation of NFAT and its translocation into the nucleus.Activation leads to induction of the diphtheria toxin A gene such thatthe cells undergo rapid cell death. This is shown using Propidium iodideas a measure of cell viability. Thus, in a large population, those cellsthat are blocked for NFAT activation by peptides that interfere with thesignaling system will survive. Serafini and colleagues used the approachto select for mutants in T cells signaling. We will use this provenNFAT-dipA systemin our peptide selections.

[0275] Again, cells will be infected as above with appropriate peptidelibraries and screened for blockade of NFAT signaling. This basicapproach, if successful, might be similarly applied to TNF-α or IL-1signaling.

[0276] There is expectation that signaling systems exist whose purposeis to provide either pro-inflammatory and anti-inflammatory signaling.As noted above, IL-4 for instance can blockade IL-6 signaling in cells.Induction of glucocorticoid expression leads to upregulation of IkB andthereby blocks NF-kB activation. Activation of anti-oxidant pathways iswell known to be similarly anti-inflammatory. Salicylate blocks NF-kBthrough regulation of cellular oxygenase levels. Although the peptidesearches outlined above might find players in such pathwaysintracellularly, we desire to search for surface molecules that mightinitiate such protective cascades.

[0277] The peptide libraries in constructs for secreted peptides andtethered peptides will be used in T cell, macrophage, and B cell systemsto select for blockade OR activation of NF-kB induction. Stimuli willinclude TNF-α and IL-1 for blockade. Activation will utilize theFACS-based systems in “reverse”. That is, we will look for peptideswhose expression leads to constitutive activation of and NF-kB reporterconstruct. In this case the reporter construct can be a TNF-a reporterdriving lacZ or GFP. The construct can similarly be IL-1 driving lacZ orGFP. For endogenous loci, we can select for cells that induce VCAM orICAM-1 expression after IL-1 signaling by FACS, both known to bepro-inflammatory responders. Again, both positive AND negative selectioncan be employed. For cells expressing tethered peptides, the selectionis straightforward as the intracellular peptides above. Post-definitionof the peptide sequence, it will be necessary to synthesize the peptidewithout the tether synthetically and determine if the peptide can workin the absence of the tether.

[0278] For secreted peptides the setup is more difficult, as theresponder cell must display the phenotype and we must trace the peptideback to the SECRETING cell. For this approach we can use any reportergene or endogenous gene in the target cells as the readout. The cells tobe infected and which will secrete the peptides will be NIH3T3. 1×10⁷3T3 cells will be infected with a fully representative library asoutlined above. Cells post-infection will be allowed to form colonies ofup to 10-20 cells. At this point media will be removed and the cellswill be overlayed with a thin layer of 0.25% agar in media. Oncesolidified, a thin, porous membrane will be placed over the cells, andwe will then overlay on this plate the responder cells at high density,also in 0.3% agar. Plates and membranes will be marked with indigoblack. In this way secreted product can diffuse to the responder cells.For selection of PRO-inflammatory secreted peptides, after 48 hoursresponder cells will be lifted from the plate on the membrane and themembrane/cells/agar will be flipped onto a correspondingly sizednitro-cellulose membrane. Cells will be lysed in situ by Sarcosyl orother appropriate detergent and then applied on the membrane to ahigh-salt solution and suction below the nitrocellulose. In this waycellular proteins will leach out of the agar matrix and bind to thenitrocellulose. The nitrocellulose can then be treated like a “Western”for induction or blockade of any of a number of different cellularproteins. In initial tests we will use reporter genes driving enzymessuch as b-gal or alkaline phosphatase to ensure assay sensitivity. As weperfect the assay it should be possible to set up direct measures ofcertain endogenous loci (such as TNF-α, NF-kB p65, etc.). Once cellareas on the membrane are noted, they can be traced back to thesecretion cells by the indigo marking of the plates and alignment.NIH-3T3 cell “patches” corresponding to the appropriate area can bepicked, expanded, and retested. As a positive control, virusesexpressing TNF-α or IL-1 will be used in initial scaled mock-ups tocalibrate the sensitivity of the search for pro-inflammatory peptides.

[0279] Similarly, one can search for blockade of pro-inflammatorysignaling. In this case, at 24 to 36 hours post plating of the respondercells, we will add a pro-inflammatory cytokine such as IL-1 or TNF-a tothe agar layers in a liquid overlaying the agar/responders. The plate isnow, from bottom to top: Secretor Cells/Membrane/Responder Cells/LiquidOverlay. The pro-inflammatory inducer will diffuse into the respondercells layer rapidly. Those cells that have been “protected” frompro-inflammatory events by a localized presence of an anti-inflammatorysecreted peptide will not respond to the stimuli. As above, these can bedetected against a background of responders by a nitrocellulose assayfor enzymatic activity. The latter, that is looking for “holes” againsta background of positivity on the nitrocellulose can be used to screenfor inhibitors of pro-inflammatory events. As a positive control,viruses expressing IL-4 will be used in initial mock-ups to calibratethe sensitivity of the search for anti-inflammatory peptides.

I claim:
 1. A method for screening for a transdominant intracellular bioactive agent capable of altering the phenotype of a cell, said method comprising the steps: a) introducing a molecular library of randomized candidate nucleic acids into a plurality of cells, wherein each of said nucleic acids comprises a different nucleotide sequence; b) screening said plurality of cells for a cell exhibiting an altered phenotype, wherein said altered phenotype is due to the presence of a transdominant bioactive agent.
 2. A method according to claim 1 further comprising the step: c) isolating said cell exhibiting an altered phenotype.
 3. A method according to claim 2 further comprising the step: d) isolating a candidate nucleic acid from said cell.
 4. A method according to claim 2 or 3 further comprising the step: e) isolating a target molecule using i) a candidate nucleic acid; or ii) the expression product of a candidate nucleic acid.
 5. A method according to claim 1 wherein said randomized candidate nucleic acids are expressed in said cells to produce a plurality of randomized candidate expression products.
 6. A method according to claim 5 wherein said randomized candidate expression products are peptides.
 7. A method according to claim 5 wherein said randomized candidate expression products are nucleic acid transcripts.
 8. A method according to claim 1 wherein said nucleic acids further comprise a presentation sequence capable of presenting said expression product in a conformationally restricted form.
 9. A method according to claim 1 wherein said introducing is with retroviral vectors.
 10. A method according to claim 1 wherein said cells are mammalian cells.
 11. A method according to claim 1 wherein said library comprises at least 10⁴ different nucleic acids.
 12. A method according to claim 1 wherein said library comprises at least 10⁵ different nucleic acids.
 13. A method according to claim 1 wherein said library comprises at least 10⁶ different nucleic acids.
 14. A method according to claim 1 wherein said library comprises at least 10⁷ different nucleic acids.
 15. A method according to claim 1 wherein said library comprises at least 10⁸ different nucleic acids.
 16. A molecular library of retroviruses comprising at least 10⁴ different randomized nucleic acids.
 17. A molecular library of retroviruses according to claim 21 comprising at least 10⁵ different randomized nucleic acids.
 18. A molecular library of retroviruses according to claim 21 comprising at least 10⁶ different randomized nucleic acids.
 19. A molecular library of retroviruses according to claim 21 comprising at least 10⁷ different randomized nucleic acids.
 20. A molecular library of retroviruses according to claim 21 comprising at least 10⁸ different randomized nucleic acids.
 21. A cellular library of mammalian cells containing a molecular library of retroviral constructs, said molecular library comprising at least 10⁴ different randomized nucleic acids.
 22. A cellular library according to claim 21 wherein said constructs are integrated into the cellular genome. 